Agrobacterium

Biotechnol Bioeng, 1999 Feb 5, 62(3), 317 - 23
Higher intracellular levels of uridinemonophosphate under nitrogen-limited conditions enhance metabolic flux of curdlan synthesis in Agrobacterium species; Kim MK et al.; Changes of intracellular nucleotide levels and their stimulatory effects on curdlan synthesis in Agrobacterium species were investigated under different culture conditions . Under nitrogen-limited conditions where curdlan synthesis was stimulated, intracellular levels of UMP were as high as 87 and those of AMP were 78 nmol/mg of cellular protein, while those under nitrogen-sufficient conditions were lower than 45 nmol/mg-protein . The levels of other nucleotides such as UDP, UTP, UDP-glucose, ADP, ATP, and ADP-glucose were lower than 30 nmol/mg-protein under both nitrogen-limited and sufficient conditions . The time profiles of curdlan synthesis and cellular nucleotide levels showed that curdlan synthesis had a positive relationship with intracellular levels of UMP and AMP . After the ammonium concentration in the medium fell below 0.1 g/L, intracellular levels of UMP and AMP increased, followed by curdlan synthesis . However, no significant changes in the specific activities of UMP kinase, UDP kinase, and UDP-glucose pyrophosphorylase were observed during cultivation . In vitro enzyme reactions for the synthesis of UDP-glucose, which serve as a precursor for curdlan synthesis, demonstrated that the synthesis of UDP-glucose increased with the increase of UMP concentration . In contrast, AMP had no effect on UDP-glucose synthesis at all . Addition of UMP in the medium increased the curdlan synthesis, whereas curdlan synthesis was inhibited in the presence of AMP . From these results, we concluded that only the higher intracellular UMP levels caused by nitrogen limitation in the medium enhance the metabolic flux of curdlan synthesis by promoting cellular UDP-glucose synthesis .

Biotechnol Bioeng, 1999 Jan 20, 62(2), 235 - 41
Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli; Wang CW et al.; The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds . Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors . To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus . The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP . These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E . coli to produce astaxanthin, an orange pigment and antioxidant . This metabolically engineered strain produces astaxanthin 50 times higher than values reported before . To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E . coli), and crtE (encoding GGPP synthase from Erwinia uredovora) . Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis . Removal of these bottlenecks results in an E . coli strain providing sufficient precursors for in vivo synthesis of isoprenoids .

Biotechnol Bioeng, 1998 Nov 5, 60(3), 375 - 84
Characterization of fluid-flow resistance in root cultures with a convective flow tubular bioreactor
Carvalho EB, Curtis WR.
Agrobacterium transformed root cultures of Hyoscyamus muticus were grown in a recirculating 2 L tubular bioreactor system . Performance of this convective flow reactor (CFR) was compared to a bubble column (BC) reactor of the same geometry: replicated CFR experiments produced an average tissue concentration of 556 +/- 4 grams fresh weight per liter in 30 d whereas the bubble column produced only 328 +/- 5 grams per liter corresponding to 25.3 +/- 0.0 and 14.3 +/- 0.5 grams dry weight per liter, respectively . Because media nutrient levels were maintained sufficiently high to saturate growth rate, the improved performance of the CFR is attributed to enhanced convective mass transfer . The pressure drops observed for flow through roots grown within the reactors were more than an order of magnitude higher than previously obtained by placing roots grown in shake culture into defined geometries . The experimentally observed flow resistance was much higher than would be predicted from correlations using the root diameter as the characteristic diameter for flow resistance . Several lines of evidence suggest that root hairs are a substantial contributor to the observed high flow resistance in these transformed root cultures . Pressure drop increased nonlinearly with velocity which could not be adequately described by a modified form of the Ergun equation . Kyan et al's (1970) equation, although predicting such curvature, relies almost exclusively on an empirical packing deflection term to describe the hydrodynamic behavior . Implications of these results to the design of submerged reactor systems for root culture are discussed .

Biotechnol Bioeng, 1998 Nov 5, 60(3), 348 - 55
Insertion of microscopic objects through plant cell walls using laser microsurgery
Buer CS, Gahagan KT, Swartzlander GA Jr, Weathers PJ.
A detailed protocol is presented for precisely inserting microscopic objects into the periplasmic region of plant callus cells using laser microsurgery . Ginkgo biloba and Agrobacterium rhizogenes were used as the model system for developing the optical tweezers and scalpel techniques using a single laser . We achieved better than 95% survival after plasmolyzing G . biloba cells, ablating a 2-4-mum hole through the cell wall using a pulsed UV laser beam, trapping and translating bacteria into the periplasmic region using a pulsed infrared laser beam, and then deplasmolyzing the cells . Insertion of bacteria is also described . A thermal model for temperature changes of trapped bacteria is included . Comparisons with other methods, such as a reverse-pressure gradient technique, are discussed and additional experiments on plants using laser microsurgery are suggested .

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3729 - 33
Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium; Ziemienowicz A et al.; Import of DNA into mammalian nuclei is generally inefficient . Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems . Here we tested whether bacterial proteins could be used to target DNA to mammalian cells . Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells . Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation . Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process . We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro . These complexes were tested for import into HeLa cell nuclei . Import of ssDNA required both VirD2 and VirE2 proteins . A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei . Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source . We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway . This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Nat Biotechnol, 1999 Mar, 17(3), 282 - 6
Iron fortification of rice seed by the soybean ferritin gene; Goto F et al.; To improve the iron content of rice, we have transferred the entire coding sequence of the soybean ferritin gene into Oryza sativa (L . cv . Kita-ake) by Agrobacterium-mediated transformation . The rice seed-storage protein glutelin promoter, GluB-1, was used to drive expression of the soybean gene specifically in developing, self-pollinated seeds (T1 seeds) of transgenic plants, as confirmed by reverse transcription PCR analysis . Stable accumulation of the ferritin subunit in the rice seed was demonstrated by western blot analysis, and its specific accumulation in the endosperm by immunologic tissue printing . The iron content of T1 seeds was as much as threefold greater than that of their untransformed counterparts.

Plant Mol Biol, 1999 Feb, 39(3), 551 - 64
A strong constitutive positive element is essential for the ammonium-regulated expression of a soybean gene encoding cytosolic glutamine synthetase; Terce-Laforgue T et al.; In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean gene GS15 encoding cytosolic glutamine synthetase, a series of 5' promoter deletions were fused to the GUS reporter gene . To allow the detection of positive and negative regulatory elements, a series of 3' deletions were fused to a -90 CaMV 35S promoter fragment placed upstream of the GUS gene . Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation . Both spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the promoter distal region is indispensable for the ammonium-regulated expression of GS15 . Interestingly, this SCPE was able to direct constitutive expression in both a legume and non-legume background to a level similar to that driven by the CaMV 35S full-length promoter . In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression . This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nodule-enhanced gene promoters . A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-specific elements . The presence of positive, negative and organ-specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA for trans-acting factor accessibility.

Plant Mol Biol, 1999 Feb, 39(3), 407 - 16
High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda); Wenck AR et al.; Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency . We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation . Additional copies of virulence genes were added to three common disarmed strains . These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542 . In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected . Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium . GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium . In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains . Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 694 - 5
Expression, crystallization and preliminary X-ray diffraction studies of N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter; Hsu WH et al.; The Agrobacterium radiobacter CCRC 14924 N-carbamyl-D-amino-acid amidohydrolase, the enzyme used for production of D-amino acids, was overexpressed in Escherichia coli JM109 . The expressed protein was crystallized by vapour diffusion using lithium sulfate as precipitant . It crystallizes in space group P21 with unit-cell parameters a = 69.8, b = 67.9 and c = 137.8 A and beta = 96.4 degrees . There are four molecules per asymmetric unit . Crystals diffract to 2.8 A resolution using a rotating-anode source at cryogenic (113 K) temperatures.

Acta Virol, 1998 Sep, 42(4), 270 - 2
Characterization of phenotype resistance to plum pox of transgenic plums expressing plum pox virus capsid gene; Ravelonandro M et al.; Resistance to plum pox virus (PPV) infection can be obtained in transgenic plants that express the virus capsid gene . An Agrobacterium-mediated transformation was used to introduce the PPV capsid gene into Prunus domestica plants . Over 11 regenerated plants (clones) were observed for the development of the disease symptoms and analysed for the presence of PPV by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) through 4 dormancy cycles . The level of protection against PPV was determined in the transformed plants, non-transformed plants, and a control transgenic plant "transformed" with the plasmid vector alone . One clone, C-5, appeared fully protected, while PT-6 and C-4 clones accumulated a low concentration of virus and the rest of the clones was entirely susceptible . Little is known about the mechanisms of resistance to virus infection in transgenic woody plants . To investigate this aspect, comparative studies based on the characteristics of resistant and susceptible clones have been started . A question, whether the phenotype resistance of clone C-5 is similar to that observed in transgenic herbaceous plants or not, has been addressed . Recent progress in this investigation is presented.

Mol Gen Genet, 1999 Feb, 261(1), 115 - 21
T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis; Bundock P et al.; The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells . The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell . Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism . The host range of A . tumefaciens is not restricted to plant species . A . tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae . In this paper we demonstrate transfer of T-DNA from A . tumefaciens to the yeast Kluyveromyces lactis . Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K . lactis . We have studied the efficiency of gene targeting at the K . lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium . We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation . Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used.

Plant J, 1998 Dec, 16(6), 735 - 43
Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana; Clough SJ et al.; The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration . In the present study, this method was evaluated and a substantially modified transformation method was developed . The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77 . Sucrose and surfactant were critical to the success of the floral dip method . Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate . Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities . Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold . Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold . Multiple ecotypes were transformable by this method . The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.

Plant J, 1998 Dec, 16(6), 673 - 80
Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions; Krizkova L et al.; Plant transformation via Agrobacterium frequently results in formation of multiple copy T-DNA arrays at one target site of the chromosome . The T-DNA copies are arranged in repeats, direct or inverted around one of the T-DNA borders . A Ti plasmid-derived transformation vector has been constructed enabling direct selection of transformants carrying at least two linked copies of T-DNA in the same orientation . The selection is based on expression of a promoterless neomycin phosphotransferase gene on one T-DNA copy from a promoter located on the other T-DNA copy . After co-cultivation of tobacco protoplasts with Agrobacterium, as many as 30% of regenerated transformed plants carried directly repeated T-DNA copies . The junction regions between two T-DNAs were amplified and 13 amplified fragments were cloned and sequenced . The involvement of T-DNA left and right border sequences in direct repeat junctions was determined . In some junctions, additional filler DNA was detected . The length of filler DNA varied from a few up to almost 300 bp . The longer filler DNAs from two clones were found to be T-DNA fragments in direct or reverse orientation . We discuss the recently suggested models for T-DNA integration and propose that the formation of direct repeats in genomes does not necessarily result from ligation of intermediates (i.e . T-strands), but more likely from the co-integration of several intermediates into one target site.

Curr Opin Plant Biol, 1998 Apr, 1(2), 161 - 5
Advances in cereal gene transfer; Komari T et al.; Over the past five years, transgenic strains of various major cereals have been produced, with transformation of rice and maize being most common . A majority of the cereal transformants obtained to date has been generated by the particle bombardment technique, but Agrobacterium-mediated transformation is rapidly becoming the method of choice . Rice, the plant in which transformation-related technology is most advanced, appears to be the model monocotyledon for basic and applied studies.

Curr Opin Microbiol, 1998 Dec, 1(6), 649 - 55
Assembly of the VirB transport complex for DNA transfer from Agrobacterium tumefaciens to plant cells; Zupan JR et al.; The VirB transporter is a type IV secretion system that mediates the genetic transformation of plant cells by Agrobacterium tumefaciens . Assembly of this transporter depends on, first, formation of a VirB7/B9 complex that stabilizes many of the VirB proteins, second, formation of a virulence-specific pilus composed primarily of VirB2 and VirB5, and, third, post-translational processing of VirB1 and VirB2.

Lett Appl Microbiol, 1999 Feb, 28(2), 137 - 41
Discrimination of Rhizobium tropici and R . leguminosarum strains by PCR-specific amplification of 16S-23S rDNA spacer region fragments and denaturing gradient gel electrophoresis (DGGE); de Oliveira VM et al.; With the aim of detecting Rhizobium species directly in the environment, specific PCR primers for Rh . tropici and Rh . leguminosarum were designed on the basis of sequence analysis of 16S-23S rDNA spacer regions of several Rh . tropici, Rh . leguminosarum and Agrobacterium rhizogenes strains . Primer specificity was checked by comparison with available rDNA spacer sequences in databases, and by PCR using DNA from target and reference strains . Sequence polymorphisms of rDNA spacer fragments among strains of the same species were detected by denaturing gradient gel electrophoresis (DGGE) . The specific PCR primers designed in this study could be applied to evaluate the diversity of Rh . tropici and Rh . leguminosarum by analysing the polymorphisms of 16S-23S spacer rDNA amplified from either whole-cell or soil-extracted DNA.

Biosci Biotechnol Biochem, 1999 Jan, 63(1), 91 - 5
Thermostability reinforcement through a combination of thermostability-related mutations of N-carbamyl-D-amino acid amidohydrolase; Ikenaka Y et al.; For the improvement of N-carbamyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of DCase from Agrobacterium sp . KNK712 was improved through various combinations of thermostability-related mutations . The thermostable temperature (defined as the temperature on heat treatment for 10 min that caused a decrease in the DCase activity of 50%) of the enzyme which had three amino acids, H57Y, P203E, and V236A, replaced was increased by about 19 degrees C . The mutant DCase, designated as 455M, was purified and its enzymatic properties were studied . The enzyme had highly increased stability against not only temperature but also pH, the optimal temperature of the enzyme being about 75 degrees C . The substrate specificity of the enzyme for various N-carbamyl-D-amino acids was changed little in comparison with that of the native enzyme . Enzymochemical parameters were also measured.

Appl Environ Microbiol, 1999 Mar, 65(3), 951 - 60
Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp . strain HR199; Overhage J et al.; Pseudomonas sp . strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth . Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis . One mutant (SK6169) was used as recipient of a Pseudomonas sp . strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58) . The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase . Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively . Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase . Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes . Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58 . Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage . Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads . In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp . strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.

Biotechniques, 1999 Feb, 26(2), 344 - 8
Chemiluminescent detection of AFLP markers; Lin JJ et al.; Nonradioactive amplified fragment-length polymorphism (AFLP) marker detection, a PCR-based, DNA-fingerprinting technique, was achieved by blotting AFLP products after electrophoresis onto a nylon membrane and subsequently hybridizing the blot with an alkaline phosphatase-labeled AFLP probe . Similar AFLP profiles were obtained by both a nonradioactive, chemiluminescent detection technique and by conventional AFLP marker detection using 32P-labeled AFLP primers . The suitability of the method using different gel systems combined with subsequent chemiluminescent detection of AFLP markers is validated by similar dendrograms that were generated using the unweighted pair group method with arithmetic averages (UPGMA) . Moreover, chemiluminescent detection of AFLP markers using a universal AFLP nonradioactive probe has been successfully applied on prokaryotes such as Agrobacterium and eukaryotic genomes such as soybean and fungi.

Gene, 1999 Feb 18, 227(2), 197 - 203
Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator; Newman JR et al.; We describe the development and analysis of broad-host-range (BHR) cloning vectors that carry the araC-PBAD controlled expression cassette from Escherichia coli . These plasmids are designed to facilitate l-arabinose-responsive control of target genes in a variety of Gram-negative bacterial hosts . BHR PBAD::lacZ fusions were used to analyze the utility of this controlled expression system in the plant pathogen Agrobacterium tumefaciens . In A . tumefaciens, the level of control afforded is significant, although less stringent than that observed in E . coli . The BHR PBAD vectors offer a useful alternative to currently used controlled expression systems, and can be employed in conjunction with other regulated promoters to simultaneously regulate expression of multiple genes . Addition of a variety of carbon sources, namely C4 acids and the anti-inducer d-fucose, allows modulation of l-arabinose induction . Activation of PBAD expression in A . tumefaciens requires a plasmid-borne copy of araC, and is not affected by endogenous regulators.

Plant Physiol, 1999 Feb, 119(2), 417 - 28
Cell-specific production and antimicrobial activity of naphthoquinones in roots of lithospermum erythrorhizon
Brigham LA, Michaels PJ, Flores HE.
Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots . Normal pigment development is limited to root hairs and root border cells in hairy roots grown on "noninducing" medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells . When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded . Acetyl-shikonin and beta-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed "hairy-root" cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested . Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots . Challenge by R . solani crude elicitor increased shikonin derivative production 30-fold . We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.

Z Naturforsch {C}, 1998 Nov-Dec, 53(11-12), 1012 - 6
Transgenic potato plants expressing soybean beta-1,3-endoglucanase gene exhibit an increased resistance to Phytophthora infestans; Borkowska M et al.; Soybean beta-1,3-endoglucanase represents a model system for studies on early plant responses to infection by fungal pathogens, and it has been implicated in the release of elicitors from fungal cell walls . In the present study, potato plants were transformed with the soybean beta-1,3-endoglucanase cDNA via Agrobacterium delivery system . The transfer of the gene into potato genome was confirmed by (i) PCR amplification, (ii) Northern blot analyses, and (iii) an increase in the activity of beta-1,3-endoglucanase in transgenic plants . The transformation resulted in an increased resistance of selected transgenic plants to infection by Phytophthora infestans, an important pathogen.

J Bacteriol, 1999 Feb, 181(3), 1049 - 53
Sporadic distribution of tRNA(Arg)CCU introns among alpha-purple bacteria: evidence for horizontal transmission and transposition of a group I intron; Paquin B et al.; A group I intron interrupts the tRNA(Arg)CCU gene of the alpha-purple bacterium Agrobacterium tumefaciens (B . Reinhold-Hurek and D . A . Shub, Nature {London} 357:173-176, 1992) . In this study, we assess the distribution of the corresponding intron among 12 additional species of alpha-purple bacteria . Of 10 newly identified tRNA(Arg)CCU genes, we found only two that contained an intron homologous to that of the Agrobacterium intron . This restricted and scattered distribution of the tRNA(Arg)CCUg intron among alpha-purple bacteria is consistent with a recent origin and horizontal transmission . Primary and secondary structural similarities between tRNA(Leu)UAA introns found in strains of the cyanobacterium Microcystis aeruginosa (K . Rudi and K . S . Jacobsen, FEMS Microbiol . Lett . 156:293-298, 1997) and alpha-purple tRNA(Arg)CCU introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA(Leu)UAA introns.

Biochemistry, 1998 Dec 22, 37(51), 18119 - 27
Kinetic mechanism of the enantioselective conversion of styrene oxide by epoxide hydrolase from Agrobacterium radiobacter AD1; Rink R et al.; Epoxide hydrolase from Agrobacterium radiobacter AD1 catalyzes the enantioselective hydrolysis of styrene oxide with an E value of 16 . The (R)-enantiomer of styrene oxide is first converted with a k(cat) of 3.8 s(-1), and the conversion of the (S)-enantiomer is inhibited . The latter is subsequently hydrolyzed with a k(cat) of 10.5 s(-1) . The pre-steady-state kinetic parameters were determined for both enantiomers with stopped-flow fluorescence and rapid-quench techniques . For (R)-styrene oxide a four-step mechanism was needed to describe the data . It involved the formation of a Michaelis complex that is in rapid equilibrium with free enzyme and substrate, followed by rapid and reversible alkylation of the enzyme . A unimolecular isomerization of the alkylated enzyme precedes the hydrolysis of the covalent intermediate, which could be observed due to an enhancement of the intrinsic protein fluorescence during this step . The conversion of (S)-styrene oxide could be described by a three-step mechanism, which also involved reversible and rapid formation of an ester intermediate from a Michaelis complex and its subsequent slow hydrolysis as the rate-limiting step . The unimolecular isomerization step has not been observed for rat microsomal epoxide hydrolase, for which a kinetic mechanism was recently established {Tzeng, H.-F., Laughlin, L . T., Lin, S., and Armstrong, R . N . (1996) J . Am . Chem . Soc . 118, 9436-9437} . For both enantiomers of styrene oxide, the Km value was much lower than the substrate binding constant K(S) due to extensive accumulation of the covalent intermediate . The enantioselectivity was more pronounced in the alkylation rates than in the rate-limiting hydrolysis steps . The combined reaction schemes for (R)- and (S)-styrene oxide gave an accurate description of the epoxide hydrolase catalyzed kinetic resolution of racemic styrene oxide.

Nat Biotechnol, 1999 Jan, 17(1), 76 - 80
High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants; Ku MS et al.; Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO2 in C4 plants into the C3 crop rice . Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein . RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene . Physiologically, the transgenic plants exhibited reduced O2 inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants . The results demonstrate a successful strategy for installing the key biochemical component of the C4 pathway of photosynthesis in C3 plants.

Mol Cells, 1998 Dec 31, 8(6), 705 - 8
Frequent occurrence of transgene deletion in transgenic plants; Kim YS et al.; The status of the transgene in tobacco plants transformed by Agrobacterium was analyzed with PCR . Twelve percent of the transgenic plants with the nptII gene showed different levels of transgene deletion, which was also found in transgenic watermelon (10-30%) and carrot (40-60%) . It appeared that the percentage of transgenic plants carrying deleted transgenes depended on both the transgene and the plant . It is suggested that the transgene should be inserted between a right border and a selection marker to reduce the number of transgenic plants containing deleted transgenes after selection.

Mol Cells, 1998 Dec 31, 8(6), 678 - 84
Cadmium resistance in transgenic tobacco plants expressing the Nicotiana glutinosa L . metallothionein-like gene; Suh MC et al.; To understand the function of metallothioneins (MTs) in plants, we introduced the Nicotiana glutinosa MT gene into tobacco (N . tabacum) plants via an Agrobacterium mediated transformation . Full-length MT cDNA was fused between the cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (nos) terminator of the pMBP1 binary vector in sense orientation . Tobacco leaf discs which were cocultivated with Agrobacterium carrying the chimeric MT gene, formed kanamycin-resistant shoots on medium containing kanamycin . The kanamycin-resistant shoots were subsequently rooted on medium containing 200 microM CdSO4 . Approximately 30% of individual transgenic plants developed normally . Nontransgenic plants promptly underwent leaf chlorosis, and their growth and development were inhibited on MS medium containing 50 microM CdSO4 . Genomic Southern blot analysis showed that the MT gene was stably integrated into the nuclear genome of transgenic tobacco plants . The expression level of MT transcripts was analyzed by RNA gel blot analysis . Self-pollinated seeds obtained from transgenic tobacco plants showing cadmium tolerance were germinated on a medium containing 100 microM CdSO4 . PCR analysis from sensitive and stably resistant T2 seedlings for cadmium sulfate confirmed a high correlation between the phenotypic expression of the MT gene and the transgenic genotype, indicating that the MT gene is inherited in the next generation.

Glycobiology, 1999 Jan, 9(1), 31 - 41
Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp . ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene; Stasinopoulos SJ et al.; Genes essential for the production of a linear, bacterial (1-->3)-beta-glucan, curdlan, have been cloned for the first time from Agrobacterium sp . ATCC31749 . The genes occurred in two, nonoverlapping, genomic fragments that complemented different sets of curdlan( crd )-deficient transposon-insertion mutations . These were detected as colonies that failed to stain with aniline blue, a (1-->3)-beta-glucan specific dye . One fragment carried a biosynthetic gene cluster (locus I) containing the putative curdlan synthase gene, crdS, and at least two other crd genes . The second fragment may contain only a single crd gene (locus II) . Determination of the DNA sequence adjacent to several locus I mutations revealed homology to known sequences only in the cases of crdS mutations . Complete sequencing of the 1623 bp crdS gene revealed highest similarities between the predicted CrdS protein (540 amino acids) and glycosyl transferases with repetitive action patterns . These include bacterial cellulose synthases (and their homologs), which form (1-->4)-beta-glucans . No similarity was detected with putative (1-->3)-beta-glucan synthases from yeasts and filamentous fungi . Whatever the determinants of the linkage specificity of these beta-glucan synthases might be, these results raise the possibility that (1-->3)-beta-glucans and (1-->4)-beta-glucans are formed by related catalytic polypeptides.

J Bacteriol, 1999 Jan, 181(2), 618 - 26
Cloning and characterization of a tetracycline resistance determinant present in Agrobacterium tumefaciens C58; Luo ZQ et al.; Agrobacterium tumefaciens C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency . We observed that a mutation affecting a tRNA processing function significantly affected the emergence of such mutants, suggesting that C58 contained a positively acting gene conferring resistance to tetracycline . A cosmid clone conferring resistance to tetracycline in Escherichia coli and Agrobacterium was isolated from a genomic bank of one such mutant . Subcloning, transposon mutagenesis, and DNA sequence analysis revealed that this DNA fragment contained two divergently transcribed genes, tetA and tetR, encoding products that were very similar to proteins of the Tet(A) class of tetracycline resistance systems . In the clone from this mutant, tetR was disrupted by an IS426 . The homologous region from wild-type NT1 contained an intact tetR gene and did not confer resistance to tetracycline . Hybridization analysis showed that of 22 members of the genus Agrobacterium surveyed, only strains C58 and T37 contained the tet determinant . Moreover, only these two strains mutated to resistance to this antibiotic . Unlike other Tet(A) systems, neither tetracycline nor a series of its derivatives induced the expression of this tet gene unit . Other polycyclic compounds, including many of plant origin, also did not induce this tet gene system . The divergent promoter region of this tet system contained a single inverted repeat element identical to one such operator repeat in the promoter region of the tet determinant from the IncP1alpha R plasmid RP4 . TetR repressor proteins from the Agrobacterium tet system and from RP4 interacted with the heterologous operators . While the repressive effect of the TetR protein from strain C58 (TetRC58) on the tetA gene from strain RP4 (tetARP4) was not relieved by tetracycline, repression of tetAC58 by TetRRP4 was lifted by this antibiotic.

Eur J Biochem, 1998 Dec 1, 258(2), 586 - 96
Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli; Tissot G et al.; Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes . In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria . We also described the cloning and sequencing of a full-length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle-transit peptide . In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain . The recombinant enzyme was isolated using an efficient three-step purification procedure . Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein . Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment . Immunocytochemistry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A . thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform . Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined . The native recombinant enzyme is a 37-kDa monomer . In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA . This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation required for enzyme activity . The recombinant enzyme was able to biotinylate efficiently apo-biotin carboxyl carrier protein (BCCP) from E . coli and apo-methylcrotonoyl-CoA carboxylase (MCCase) from A . thaliana . Apparent Km values for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 130 nM, 4.4 microM and 32 microM, respectively . Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end-products of the enzyme-catalysed reaction . Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.

Mol Gen Genet, 1998 Nov, 260(4), 362 - 71
Effect of ploidy and homozygosity on transgene expression in primary tobacco transformants and their androgenetic progenies; Beaujean A et al.; Expression of a transgene is rarely analysed in the androgenetic progenies of the transgenic plants . Here, we report differential transgene expression in androgenetic haploid and doubled haploid (DH) tobacco plants as compared to the diploid parental lines, thus demonstrating a gene dosage effect . Using Agrobacterium-mediated transformation, and bacterial reporter genes encoding neomycin phosphotransferase (nptII) and beta-glucuronidase (uidA/ GUS), driven respectively by the mas 1' and mas 2' promoters, we have generated more than 150 independent transgenic (R0) Nicotiana tabacum plants containing one or more T-DNA copies . Transgene analyses of these R0, their selfed R1 lines and their corresponding haploid progenies showed an obvious position effect (site of T-DNA insertion on chromosome) on uidA expression . However, transgene (GUS) expression levels were not proportional to transgene copy number . More than 150 haploids and doubled haploids, induced by treatment with colchicine, were produced from 20 independent transgenic R0 plants containing single and multiple copies of the uidA gene . We observed that homozygous DH plants expressed GUS at approximately 2.9-fold the level of the corresponding parental haploid plants . This increase in transgene expression may be attributed mainly to the increase (2-fold) in chromosome number . Based on this observation, we suggest a strong link between chromosome number (ploidy dosage effect) and transgene expression . In particular, we demonstrate the effect on its expression level of converting the transgene from the heterozygous (in R0 plants) to the homozygous (DH) state: e.g . an increase of 50% was observed in the homozygous DH as compared to the original heterozygous diploid plants . We propose that ploidy coupled with homozygosity can result in a new type of gene activation, creating differences in gene expression patterns.

Plant Mol Biol, 1998 Dec, 38(6), 1021 - 9
Identification of class B and class C floral organ identity genes from rice plants; Kang HG et al.; The functions of two rice MADS-box genes were studied by the loss-of-function approach . The first gene, OsMADS4, shows a significant homology to members in the PISTILLATA (PI) family, which is required to specify petal and stamen identity . The second gene, OsMADS3, is highly homologous to the members in the AGAMOUS (AG) family that is essential for the normal development of the internal two whorls, the stamen and carpel, of the flower . These two rice MADS box cDNA clones were connected to the maize ubiquitin promoter in an antisense orientation and the fusion molecules were introduced to rice plants by the Agrobacterium-mediated transformation method . Transgenic plants expressing antisense OsMADS4 displayed alterations of the second and third whorls . The second-whorl lodicules, which are equivalent to the petals of dicot plants in grasses, were altered into palea/lemma-like organs, and the third whorl stamens were changed to carpel-like organs . Loss-of-function analysis of OsMADS3 showed alterations in the third and fourth whorls . In the third whorl, the filaments of the transgenic plants were changed into thick and fleshy bodies, similar to lodicules . Rather than making a carpel, the fourth whorl produced several abnormal flowers . These phenotypes are similar to those of the agamous and plena mutants in Arabidopsis and Antirrhinum, respectively . These results suggest that OsMADS4 belongs to the class B gene family and OsMADS3 belongs to the class C gene family of floral organ identity determination.

J Bacteriol, 1999 Jan, 181(1), 186 - 96
pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export; Lee LY et al.; When coresident with the Ti (tumor-inducing) plasmid, the 21-kDa product of the osa gene of the plasmid pSa can suppress crown gall tumorigenesis incited by Agrobacterium tumefaciens . Neither T-DNA processing nor vir (virulence) gene induction is affected by the presence of osa in the bacterium . We used Arabidopsis thaliana root segments and tobacco leaf discs to demonstrate that Osa inhibits A . tumefaciens from transforming these plants to the stable phenotypes of tumorigenesis, kanamycin resistance, and stable beta-glucuronidase (GUS) expression . When A . tumefaciens contained osa, the lack of expression of transient GUS activity in infected plant tissues, as well as the lack of systemic viral symptoms following agroinfection of Nicotiana benthamiana by tomato mottle virus, suggested that oncogenic suppression by Osa occurs before T-DNA enters the plant nucleus . The extracellular complementation of an A . tumefaciens virE2 mutant (the T-DNA donor strain) by an A . tumefaciens strain lacking T-DNA but containing a wild-type virE2 gene (the VirE2 donor strain) was blocked when osa was present in the VirE2 donor strain, but not when osa was present in the T-DNA donor strain . These data indicate that osa inhibits VirE2 protein, but not T-DNA export from A . tumefaciens . These data further suggest that VirE2 protein and T-DNA are separately exported from the bacterium . The successful infection of Datura stramonium plants and leaf discs of transgenic tobacco plants expressing VirE2 protein by an A . tumefaciens virE2 mutant carrying osa confirmed that oncogenic suppression by osa does not occur by blocking T-DNA transfer . Overexpression of virB9, virB10, and virB11 in A . tumefaciens did not overcome oncogenic suppression by osa . The finding that the expression of the osa gene by itself, rather than the formation of a conjugal intermediate with pSa, blocks transformation suggests that the mechanism of oncogenic suppression by osa may differ from that of the IncQ plasmid RSF1010.

Plant Mol Biol, 1998 Nov, 38(5), 743 - 53
A 42 bp fragment of the pmas1' promoter containing an ocs-like element confers a developmental, wound- and chemically inducible expression pattern; Guevara-Garcia A et al.; Synthesis of mannopine in plant tissues infected with Agrobacterium tumefaciens is controlled by a divergent promoter (pmas2' and pmas1') that in 479 bp contains all the cis-acting elements necessary to direct tissue-specific and wound-inducible expression . In this report, using transgenic tobacco plants harboring a pmas1'-beta-glucuronidase (GUS) gene fusion, we investigated the developmental expression pattern directed by pmas1' in the early stages of development and the responses of pmas1' to different chemical inducers . It was found that this promoter can respond to auxins, cytokinins, methyl jasmonate (MJ), salicylic acid (SA) and its analogue 2,6-dichloroisonicotinic acid (iNA) . Treatment with chemical inducers also showed that the effects of iNA are organ-dependent, that wound-induction is a complex response mediated by at least two different chemical signals, and that MJ stimulates changes in the tissue-specific and developmental expression pattern directed by the ptmas1' promoter . Using chimeric promoters we demonstrate that an ocs-like element (ocs+1) directs MJ responses in an orientation-dependent manner and that sequences around the ocs+1 are important to maintain the inducible and developmental properties of this cis-regulatory element.

J Bacteriol, 1998 Dec, 180(24), 6597 - 606
Stability of the Agrobacterium tumefaciens VirB10 protein is modulated by growth temperature and periplasmic osmoadaption; Banta LM et al.; Export of oncogenic T-DNA from the phytopathogen Agrobacterium tumefaciens is mediated by the products of the virB operon . It has recently been reported (K . J . Fullner and E . W . Nester, J . Bacteriol . 178:1498-1504, 1996) that DNA transfer does not occur at elevated temperatures; these observations correlate well with much earlier studies on the temperature sensitivity of crown gall tumor development on plants . In testing the hypothesis that this loss of DNA movement reflects a defect in assembly or maintenance of a stable DNA transfer machinery at high temperature, we have found that steady-state levels of VirB10 are sensitive to growth temperature while levels of several other VirB proteins are considerably less affected . This temperature-dependent failure to accumulate VirB10 is exacerbated in an attachment-deficient mutant strain (chvB) which exhibits pleiotropic defects in periplasmic osmoadaption, and virulence of a chvB mutant can be partially restored by lowering the temperature at which the bacteria and the plant tissue are cocultivated . Furthermore, the stability of VirB10 is diminished in cells lacking functional VirB9, but only under conditions of low osmolarity . We propose that newly synthesized VirB10 is inherently labile in the presence of a large osmotic gradient across the inner membrane and is rapidly degraded unless it is stabilized by VirB9-dependent assembly into oligomeric complexes . The possibility that VirB10-containing complexes are not assembled properly at elevated temperatures suggests an explanation for the decades-old observation that tumor formation is exquisitely sensitive to ambient temperature.

J Bacteriol, 1998 Dec, 180(24), 6557 - 64
Gene organization and transcription analysis of the Agrobacterium tumefaciens glycogen (glg) operon: two transcripts for the single phosphoglucomutase gene; Ugalde JE et al.; The gene organization and transcription of the Agrobacterium glg operon differ from those in other bacteria . Agrobacterium tumefaciens A348 contains a 9.1-kb gene cluster harboring genes for glycogen metabolism . The nucleotide sequence and gene organization of a region containing ADP-glucose pyrophosphorylase (glgC), glycogen synthetase (glgA), and phosphoglucomutase (pgm) genes have been previously described (A . Uttaro and R . A . Ugalde, Gene 150:117-122, 1994) . In this work we report that the glycogen phosphorylase (glgP) and branching enzyme (glgB) genes are located immediately upstream of this region . The complete nucleotide sequences of the glgP and glgB genes were obtained, and mutants were constructed by targeted insertional mutagenesis with a kanamycin cassette . Enzymatic assays and reverse transcription PCR carried out with the wild type and with glgP and glgB mutants, as well as primer extension experiments and beta-galactosidase fusions, revealed that this region containing five open reading frames (glgPBCA and pgm) is transcribed unidirectionally as a single operon under the control of a promoter located upstream of the glycogen phosphorylase gene (glgP) . An alternative transcript was identified starting 168 bp upstream of an internal ATG start codon of the pgm gene, which is translated as a 71-amino-acid-shorter Pgm protein which complements in vivo a pgm mutant . This alternative transcript has a promoter with the motif TATCAAN5G, identified in octopine Ti plasmid as an autoinducible TraR promoter . This promoter is >200 times more efficient in A . tumefaciens than in Escherichia coli, as judged by the level of enzymatic activity of a lacZ-pgm fusion.

Microbiology, 1998 Nov, 144 ( Pt 11), 3149 - 57
Molecular and functional analysis of pTAV320, a repABC-type replicon of the Paracoccus versutus composite plasmid pTAV1; Bartosik D et al.; The second replicator region of the native plasmid pTAV1 of Paracoccus versutus has been identified thus proving the composite nature of this replicon . The minimal replicon designated pTAV320 (4.3 kb) was cloned and sequenced . pTAV320 encodes three putative proteins--RepA, RepB and RepC . This replicator region shows strong structural and functional similarity to repABC-type replicons found in several Agrobacterium and Rhizobium plasmids . The origin of replication appears to be localized within the coding sequence of the repC gene . RepC was shown to be essential for replication . RepA and RepB were necessary for stable maintenance of the plasmid, which implies a role in active partitioning . The presence of the complete sequence of pTAV320 (in its non-replicative form) could stabilize in cis pTAV202, a mini-replicon derived from the other pTAV1 replicator region . Deletions introduced into the repC gene abolished the 'stabilizing' activity of pTAV320, suggesting that the centromere-like sequence, necessary for partitioning, might be localized within this gene . The two replicator regions of pTAV1 (pTAV320 and pTAV202) expressed incompatibility towards the parental plasmid but were compatible in trans in P . versutus cells . The pTAV320 replicon can be maintained in several Paracoccus, Agrobacterium, Rhizobium and Rhodobacter strains in addition to P . versutus.

Biosci Biotechnol Biochem, 1998 Oct, 62(10), 1839 - 44
Immobilization of N-carbamyl-D-amino acid amidohydrolase; Nanba H et al.; N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp . strain KNK712, has been immobilized for use in the production of D-amino acids . The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme . The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively . DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite . The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme . After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively . DCase produced with Pseudomonas sp . strain KNK003A and Pseudomonas sp . strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp . strain KNK712 were also immobilized on Duolite A-568 . The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp . strain KNK712, though the activity was lower than that of strain KNK712.

Appl Environ Microbiol, 1998 Dec, 64(12), 4912 - 7
Genetic diversity and phylogeny of rhizobia that nodulate acacia spp . in morocco assessed by analysis of rRNA genes
Khbaya B, Neyra M, Normand P, Zerhari K, Filali-Maltouf A.
Forty rhizobia nodulating four Acacia species (A . gummifera, A . raddiana, A . cyanophylla, and A . horrida) were isolated from different sites in Morocco . These rhizobia were compared by analyzing both the 16S rRNA gene (rDNA) and the 16S-23S rRNA spacer by PCR with restriction fragment length polymorphism (RFLP) analysis . Analysis of the length of 16S-23S spacer showed a considerable diversity within these microsymbionts, but RFLP analysis of the amplified spacer revealed no additional heterogeneity . Three clusters were identified when 16S rDNA analysis was carried out . Two of these clusters include some isolates which nodulate, nonspecifically, the four Acacia species . These clusters, A and B, fit within the Sinorhizobium lineage and are closely related to S . meliloti and S . fredii, respectively . The third cluster appeared to belong to the Agrobacterium-Rhizobium galegae phylum and is more closely related to the Agrobacterium tumefaciens species . These relations were confirmed by sequencing a representative strain from each cluster.

FEMS Microbiol Lett, 1998 Nov 15, 168(2), 297 - 301
Localization of the protein VirB1 involved in contact formation during conjugation among Agrobacterium cells; Chumakov MI et al.; Supramembrane structures of Agrobacterium, which link cells during mating, were for the first time visualized using transmission electron microscopy . The initial cell contact was found to be mediated by long pili . Using colloidal gold-labeled, VirB1-specific antibodies, it was established that VirB1 proteins enter into the composition of short pilus-like structures, which emerge at the poles of acetosyringone (AS)-induced agrobacterial cells . Labeling of non-centrifuged agrobacterial cells on a nitrocellulose membrane using colloidal gold-conjugated antibodies to VirB1 showed that the labeled complex could bind to AS-induced cells, but failed to form red stains during incubation with cells of the Ti plasmidless A . tumefaciens strains LBA288 and UBAPF-2.

Can J Microbiol, 1998 Aug, 44(8), 743 - 52
Characteristics of phenanthrene-degrading bacteria isolated from soils contaminated with polycyclic aromatic hydrocarbons; Aitken MD et al.; Ten bacterial strains were isolated from seven contaminated soils by enrichment with phenanthrene as the sole carbon source . These isolates and another phenanthrene-degrading strain were examined for various characteristics related to phenanthrene degradation and their ability to metabolize 12 other polycyclic aromatic hydrocarbons (PAH), ranging in size from two to five rings, after growth in the presence of phenanthrene . Fatty acid methyl ester analysis indicated that at least five genera (Agrobacterium, Bacillus, Burkholderia, Pseudomonas, and Sphingomonas) and at least three species of Pseudomonas were represented in this collection . All of the strains oxidized phenanthrene according to Michaelis-Menten kinetics, with half-saturation coefficients well below the aqueous solubility of phenanthrene in all cases . All but one of the strains oxidized 1-hydroxy-2-naphthoate following growth on phenanthrene, and all oxidized at least one downstream intermediate from either or both of the known phenanthrene degradation pathways . All of the isolates could metabolize (oxidize, mineralize, or remove from solution) a broad range of PAH, although the exact range and extent of metabolism for a given substrate were unique to the particular isolate . Benz{a}anthracene, chrysene, and benzo{a}pyrene were each mineralized by eight of the strains, while pyrene was not mineralized by any . Pyrene was, however, removed from solution by all of the isolates, and the presence of at least one significant metabolite from pyrene was observed by radiochromatography for the five strains in which such metabolites were sought . Our results support earlier indications that the mineralization of pyrene by bacteria may require unique metabolic capabilities that do not appear to overlap with the determinants for mineralization of phenanthrene or other high molecular weight PAH.

J Bacteriol, 1998 Dec, 180(23), 6325 - 31
Mutational analysis of the Rhizobium etli recA operator; Tapias A et al.; Based upon our earlier studies (A . Tapias, A . R . Fernandez de Henestrosa, and J . Barbe, J . Bacteriol . 179:1573-1579, 1997) we hypothesized that the regulatory sequence of the Rhizobium etli recA gene was TTGN11CAA . However, further detailed analysis of the R . etli recA operator described in the present work suggests that it may in fact be GAACN7GTAC . This new conclusion is based upon PCR mutagenesis analysis carried out in the R . etli recA operator, which indicates that the GAAC and GTAC submotifs found in the sequence GAACN7GTAC are required for the maximal stimulation of in vivo transcription and in vitro DNA-protein complex formation . This DNA-protein complex is also detected when the GAACN7GTAC wild-type sequence is modified to obtain GAACN7GAAC, GTACN7GTAC, or GAACN7GTTC . The wild-type promoters of the Rhizobium meliloti and Agrobacterium tumefaciens recA genes, which also contain the GAACN7GTAC sequence, compete with the R . etli recA promoter for the DNA-protein complex formation but not with mutant derivatives in any of these motifs, indicating that the R . etli, R . meliloti, and A . tumefaciens recA genes present the same regulatory sequence.

J Bacteriol, 1998 Dec, 180(23), 6164 - 72
Genetic and sequence analysis of the pTiC58 trb locus, encoding a mating-pair formation system related to members of the type IV secretion family; Li PL et al.; Conjugal transfer of pTiC58 requires two regions, tra which contains the oriT and several genes involved in DNA processing and a region of undefined size and function that is located at the 2-o'clock position of the plasmid . Using transposon mutagenesis with Tn3HoHo1 and a binary transfer system, we delimited this second region, called trb, to an 11-kb interval between the loci for vegetative replication and nopaline catabolism . DNA sequence analysis of this region identified 13 significant open reading frames (ORFs) spanning 11,003 bp . The first, encoding traI, already has been described and is responsible for the synthesis of Agrobacterium autoinducer (AAI) (I . Hwang, P.-L . Li, L . Zhang, K . R . Piper, D . M . Cook, M . E . Tate, and S . K . Farrand, Proc . Natl . Acad . Sci . USA 91:4639-4643, 1994) . Translation products of the next 11 ORFs showed similarities to those of trbB, -C, -D, -E, -J, -K, -L, -F, -G, -H, and -I of the trb region of the octopine-type Ti plasmid pTi15955 and of the tra2 core region of RP4 . In RP4, these genes encode mating-pair formation functions and are essential for the conjugal transfer of the IncP plasmid . Each of the trb gene homologues is oriented counterclockwise on the Ti plasmid . Expression of these genes, as measured by using the lacZ fusions formed by Tn3HoHo1, required the traI promoter and the transcriptional activator TraR along with its coinducer, AAI . While related to that of RP4, the trb system of pTiC58 did not allow propagation of the trb-specific bacteriophages PRD1, PRR1, and Pf3 . The products of several trb genes of the Ti plasmid are similar to those of other loci that encode DNA transfer or protein secretion systems, all of which are members of the type IV secretion family.

Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1277 - 90
Allorhizobium undicola gen . nov., sp . nov., nitrogen-fixing bacteria that efficiently nodulate Neptunia natans in Senegal; de Lajudie P et al.; A group of nodule isolates from Neptunia natans, an indigenous stemnodulated tropical legume found in waterlogged areas of Senegal, was studied . Polyphasic taxonomy was performed, including SDS-PAGE of total proteins, auxanography using API galleries, host-plant specificity, PCR-RFLP of the internal transcribed spacer region between the 16S and the 23S rRNA coding genes, 16S rRNA gene sequencing and DNA-DNA hybridization . It was demonstrated that this group is phenotypically and phylogenetically separate from the known species of Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, Bradyrhizobium and Azorhizobium . Its closest phylogenetic neighbour, as deduced by 16S rRNA gene sequencing, is Agrobacterium vitis (96.2% sequence homology) . The name Allorhizobium undicola gen . nov., sp . nov., is proposed for this group of bacteria, which are capable of efficient nitrogen-fixing symbiosis with Neptunia natans, and the type strain is ORS 992T (= LMG 11875T).

Mol Gen Genet, 1998 Oct, 259(6), 559 - 68
A tobacco homologue of the Ri-plasmid orf13 gene causes cell proliferation in carrot root discs; Frundt C et al.; The tobacco genome contains genes, called cellular rol (c-rol) genes, that are very similar in sequence to genes present in the T-DNA of the Agrobacterium rhizogenes Ri-plasmid . We have cloned two homologues (torf13-1 and torf13-2) of the Ri-plasmid orf13 gene from Nicotiana tabacum L . cv . Havana 425 . The clone torf13-1 has a 594-bp open reading frame (ORF) which is similar in sequence (77-82% for DNA and 67-77% for the deduced amino acid sequence) to orf13 genes of the agropine, mikimopine, and mannopine Ri-plasmids and the N . glauca homologue Ngorf13 . Southern analyses showed that there are at least two torf13 genes derived from the N . tomentosiformis ancestor of tobacco, strongly suggesting that torf13 resulted from an ancient transfer between ancestors of modern A . rhizogenes and tobacco . Steady-state expression of torf13 mRNA is high in sepals, petals, shoot tips and in younger leaves, but considerably lower in stem tissues, lower leaves and roots . Treatment of cultured leaf discs for 5-20 days on medium containing auxin (10.7 microM alpha-naphthaleneacetic acid) and cytokinin (1.4 microM kinetin) resulted in a marked down-regulation of torf13 mRNA accumulation . Therefore, torf13 is transcriptionally active in normal tobacco tissues and the steady-state mRNA level is regulated . Inoculation of carrot-root discs with A . tumefaciens strains carrying the mannopine Ri-plasmid orf13 and torf13-1 regulated by the strong cauliflower mosaic virus 35S RNA promoter induced the formation of dense green callus on the disc surface . These findings indicate that at least one function of the orf13 ORF is conserved in the tobacco homologue, and provide direct evidence that a c-rol gene can influence cell proliferation.

Mol Plant Microbe Interact, 1998 Nov, 11(11), 1136 - 41
Agrobacterium tumefaciens transformation of the radiation hypersensitive Arabidopsis thaliana mutants uvh1 and rad5; Nam J et al.; The Arabidopsis thaliana mutants uvh1 and rad5, originally identified as radiation hypersensitive, were reported to be deficient in T-DNA integration based on the relative efficiencies of stable transformation and T-DNA transfer . We reassessed these mutants for susceptibility to transformation by Agrobacterium tumefaciens . The mutant rad5 showed a significant reduction in the efficiency of transient as well as stable transformation, compared with its wild-type progenitor . These data indicate that rad5 is blocked at a step in the transformation process prior to T-DNA integration . We additionally found, using both an in vitro root inoculation and an in vivo flower bolt inoculation assay, that the mutant uvh1 is as susceptible to A . tumefaciens-mediated transformation as is its wild-type progenitor, C10.

Mol Plant Microbe Interact, 1998 Nov, 11(11), 1119 - 29
Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria; Cha C et al.; Many gram-negative bacteria regulate expression of specialized gene sets in response to population density . This regulatory mechanism, called autoinduction or quorum-sensing, is based on the production by the bacteria of a small, diffusible signal molecule called the autoinducer . In the most well-studied systems the autoinducers are N-acylated derivatives of L-homoserine lactone (acyl-HSL) . Signal specificity is conferred by the length, and the nature of the substitution at C-3, of the acyl side-chain . We evaluated four acyl-HSL bioreporters, based on tra of Agrobacterium tumefaciens, lux of Vibrio fischeri, las of Pseudomonas aeruginosa, and pigment production by Chromobacterium violaceum, for their ability to detect sets of 3-oxo acyl-HSLs, 3-hydroxy acyl-HSLs, and alkanoyl-HSLs with chain lengths ranging from C4 to C12 . The traG::lacZ fusion reporter from the A . tumefaciens Ti plasmid was the single most sensitive and versatile detector of the four . Using this reporter, we screened 106 isolates representing seven genera of bacteria that associate with plants . Most of the Agrobacterium, Rhizobium, and Pantoea isolates and about half of the Erwinia and Pseudomonas isolates gave positive reactions . Only a few isolates of Xanthomonas produced a detectable signal . We characterized the acyl-HSLs produced by a subset of the isolates by thin-layer chromatography . Among the pseudomonads and erwinias, most produced a single dominant activity chromatographing with the properties of N-(3-oxo-hexanoyl)-L-HSL . However, a few of the erwinias, and the P . fluorescens and Ralstonia solanacearum isolates, produced quite different signals, including 3-hydroxy forms, as well as active compounds that chromatographed with properties unlike any of our standards . The few positive xanthomonas, and almost all of the agrobacteria, produced small amounts of a compound with the chromatographic properties of N-(3-oxo-octanoyl)-L-HSL . Members of the genus Rhizobium showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals . Several isolates produced extremely nonpolar compounds indicative of very long acyl side-chains . Production of these compounds suggests that quorum-sensing is common as a gene regulatory mechanism among gram-negative plant-associated bacteria.

Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1672 - 5
Relationship between an increase in thermostability and amino acid substitutions in N-carbamyl-D-amino acid amidohydrolase; Ikenaka Y et al.; For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp . KNK712 was mutagenized to increase its enzymatic thermostability . In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10 degrees C increase in thermostability . These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.

Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1668 - 71
Increase in thermostability of N-carbamyl-D-amino acid amidohydrolase on amino acid substitutions; Ikenaka Y et al.; To improve the production of D-amino acids using an immobilized N-carbamyl-D-amino acid amidohydrolase, the enzyme gene of Agrobacterium sp . KNK712 was mutagenized randomly to increase its thermostability . The gene was inserted into M13mp19, mutagenized with hydroxylamine, ligated into pUC19 after restriction endonuclease digestion, and then used to transform Escherichia coli . The resultant transformants were screened by a newly developed colorimetric enzyme assay method, and the candidate colonies corresponding to red spots were separated from the master plates . Using cell-free extracts of these clones, the properties of the enzymes produced were investigated, it being proved that these enzymes had almost the same activity and improved thermostability by about 5 degrees C compared with those of the native enzyme . As found on enzyme gene analysis of these mutants, the 57th amino acid, histidine, of the enzyme was changed to tyrosine, or the 203rd amino acid, proline, to leucine or serine.

Appl Environ Microbiol, 1998 Nov, 64(11), 4566 - 72
Engineering of a single-chain variable-fragment (scFv) antibody specific for the stolbur phytoplasma (Mollicute) and its expression in escherichia coli and tobacco plants
Le Gall F, Bove JM, Garnier M.
From a hybridoma cell line (2A10) producing an immunoglobulin G1 directed against the major membrane protein of the stolbur phytoplasma, we have engineered scFv (single-chain variable-fragment) antibodies from the variable heavy (VH) and light (VL) domains of the immunoglobulin . The scFv gene was cloned and expressed in Escherichia coli . The expressed protein of 30 kDa could be recovered from the periplasmic fraction of the bacterial cells and was shown to be fully functional toward its phytoplasmal antigen, since enzyme-linked immunosorbent assay or immunofluorescence (IF) detection of the stolbur phytoplasma antigen by the scFv was identical to that of the native immunoglobulin . The scFv gene was then cloned in plasmid pBG-dAb-BIN of Agrobacterium tumefaciens to transform tobacco plants . The transformed plants were screened by PCR and Northern blotting for the presence and expression of the transgene, respectively, and by IF for expression of the scFv . One transgenic tobacco line, 1A6, was selected for challenge inoculation with the stolbur phytoplasma . When grafted on a stolbur phytoplasma-infected tobacco rootstock, the transgenic tobacco shoots grew free of symptoms and flowered after 2 months, while normal tobacco shoots showed severe stolbur symptoms during the same period and eventually died.

J Bacteriol, 1998 Nov, 180(21), 5660 - 7
Wound-released chemical signals may elicit multiple responses from an Agrobacterium tumefaciens strain containing an octopine-type Ti plasmid; Kalogeraki VS et al.; The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells . Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope . To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci . One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid . The virH operon contains two genes that resemble P-450-type monooxygenases . The other locus encodes a 0.5-kb gene designated virK . In addition, we identified other potential genes in this region that are not conserved between these two plasmids . To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ . Expression of these genes was also measured by nuclease S1 protection assays . virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone . Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanchoe diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.

J Bacteriol, 1998 Nov, 180(21), 5632 - 8
The phenolic recognition profiles of the Agrobacterium tumefaciens VirA protein are broadened by a high level of the sugar binding protein ChvE; Peng WT et al.; The formation of crown gall tumors by Agrobacterium tumefaciens requires that the virulence (vir) genes be induced by chemical signals which consist of specific phenolic compounds and monosaccharides, synthesized at plant wound sites . Signal transduction in the activation of these genes is mediated by the VirA-VirG two-component regulatory system, together with ChvE, a glucose-galactose binding protein which interacts with VirA . We have previously presented genetic evidence that virA senses phenolic compounds directly (Y.-W . Lee, S . Jin, W.-S . Sim, and E . W . Nester, Proc . Natl . Acad . Sci . USA 92:12245-12249, 1995) . The vir genes of strain KU12 can be induced by 4-hydroxyacetophenone, p-coumaric acid, and phenol, whereas these same phenolic compounds are weak inducers of the vir genes of strain A6 . In this report, we show that a specific inducing sugar can broaden the specificity of the phenolic compound which VirA senses . 4-Hydroxyacetophenone and other related phenolic compounds function as inducing phenolic compounds with the virA gene of A6 if arabinose replaces glucose as the inducing sugar . We further demonstrate that this broadened specificity for phenolic inducers results from the increased level of ChvE through induction by arabinose via the regulatory protein GbpR . If high levels of ChvE are present, then poorly inducing phenolic compounds can induce the vir genes to high levels in combination with glucose . Comparing the induction response of the wild type and that of a VirA mutant with a mutation in its receiver domain revealed that the activity of the receiver domain is controlled by the periplasmic domain . We discuss these observations in terms of how VirA senses and transduces signals elicited by the two classes of plant signal molecules.

Mol Gen Genet, 1998 Sep, 259(4), 414 - 23
Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus; Schauser L et al.; Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia . The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome . Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus . The sym8 mutants are arrested during infection by the bacteria early in the developmental process . The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed . These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L . japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti . Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles . In the screen reported here independent symbiotic loci thus appeared with a frequency of approximately 1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.

Mol Cell Probes, 1998 Oct, 12(5), 259 - 62
The construction and use of a PCR internal control; Sachadyn P et al.; An example of the application and contruction of a polymerase chain reaction (PCR) internal control is presented . The internal control is synthesized in one PCR reaction . The primers used in this reaction possess 5' over-hanging ends which are identical to the primers used in the diagnostic reaction, whereas their 3' ends are complementary to a predetermined DNA sequence (pUC19 in this case) of defined length and sequence . As the sequence of the control except for primer sites, is not homologous to the PCR signal product, the formation of heteroduplexes and non-specific PCR products should not occur . Neither is there a risk that the target DNA will contaminate the internal control . However, the simultaneous amplification of two different DNA fragments flanked by the same primer sites resulted in either inhibition or enhancement of one or both products depending on the molar ratio of those DNA fragments . The presented method may be applied to construction of internal controls for quantitative PCR . The internal control was developed and tested for use in a PCR detection system for Agrobacterium tumefaciens .

Genetika, 1998 Aug, 34(8), 1056 - 62
{Formation of deletional derivatives of the Ti-plasmid pGV3850 in a conjugative transfer from Agrobacterium tumefaciens to Escherichia coli}; Velikov VA et al.; The process of the transfer of the Ti-plasmid vector pGV3850 with the plasmid pBR322 inserted into the T-DNA region from Agrobacterium tumefaciens to a non-plasmid strain of Escherichia coli was studied . The transferred Ti-plasmid was found to be exposed to deletions and formed a wide range of derivatives with a size ranging from 3-4 kb to 50 kb, maintained in E . coli due to ColE1-replicon . The Ti-plasmid is also inserted into the chromosome of the recipient bacterium with at least a 100-fold lower frequency than the formation of deletional derivatives . It was shown that the induction of vir genes controlling the transfer of T-DNA into plants has no appreciable effect on the efficiency of obtaining transconjugates in mating with E . coli . The deletion of the genetic material of megaplasmids with the inserted functional site OriV ColE1, as a result of the conjugative transfer from cells of different bacteria to the cells of E . coli, was proposed for molecular cloning.

Braz J Med Biol Res, 1998 Aug, 31(8), 1095 - 8
The radioprotective effect of a new aminothiol (20-PRA); Dolabela MF et al.; We examined the radioprotective effect of aminothiol 2-N-propylamine-cyclo-hexanethiol (20-PRA) on a human leukemic cell line (K562) following various radiation doses (5, 7.5 and 20 Gy) using a source of 60Co gamma-rays . At 5 Gy and 1 nM 20-PRA, a substantial protective effect (58%) was seen 24 h after irradiation, followed by a decrease at 48 h (11%) . At the high radiation dose (20 Gy) a low protective effect was also seen (35%) . In addition, the antitumorigenic potential of 10 nM 20-PRA was shown by the inhibition of crown gall formation induced by Agrobacterium tumefaciens . The radioprotective potency of 20-PRA is 10(5)-10(6) times higher than that of the aminothiol WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) whose protective effect is in the 0.1 to 1.0 mM range.

J Clin Microbiol, 1998 Nov, 36(11), 3217 - 22
Identification of Brucella by ribosomal-spacer-region PCR and differentiation of Brucella canis from other Brucella spp . pathogenic for humans by carbohydrate profiles; Fox KF et al.; Molecular and chemical characteristics often provide complementary information in the differentiation of closely related organisms . The genus Brucella consists of a highly conserved group of organisms . Identification of the four species pathogenic in humans (Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis) is problematic for many clinical laboratories that depend primarily on serology and phenotypic characteristics to differentiate species . PCR amplification of the 16S-23S ribosomal DNA interspace region was evaluated for species-specific polymorphism . B . abortus, B . melitensis, B . suis, and B . canis produced identical PCR interspace profiles . However, these PCR products were unique to brucellae, allowing them to be readily distinguished from other gram-negative bacteria (including Bartonella spp . and Agrobacterium spp.) . Carbohydrate profiles differentiated B . canis from the other three Brucella species due to the absence of the rare amino sugar quinovosamine in the three other species . PCR of the rRNA interspace region is useful in identification of the genus Brucella, while carbohydrate profiling is capable of differentiating B . canis from the other Brucella species.

EMBO J, 1998 Oct 15, 17(20), 6086 - 95
Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells; Salomon S et al.; To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene . After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker . Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break . Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions . This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation . Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site . This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome.

Plant Physiol, 1998 Oct, 118(2), 543 - 50
Natural genetic transformation by agrobacterium rhizogenes . Annual flowering in two biennials, belgian endive and carrot
Limami MA, Sun LY, Douat C, Helgeson J, Tepfer D.
Genetic transformation of Belgian endive (Cichorium intybus) and carrot (Daucus carota) by Agrobacterium rhizogenes resulted in a transformed phenotype, including annual flowering . Back-crossing of transformed (R1) endive plants produced a line that retained annual flowering in the absence of the other traits associated with A . rhizogenes transformation . Annualism was correlated with the segregation of a truncated transferred DNA (T-DNA) insertion . During vegetative growth, carbohydrate reserves accumulated normally in these annuals, and they were properly mobilized prior to anthesis . The effects of individual root-inducing left-hand T-DNA genes on flowering were tested in carrot, in which rolC (root locus) was the primary promoter of annualism and rolD caused extreme dwarfism . We discuss the possible adaptive significance of this attenuation of the phenotypic effects of root-inducing left-hand T-DNA.

Gene, 1998 Oct 5, 220(1-2), 83 - 9
A chemotaxis cluster from Agrobacterium tumefaciens; Wright EL et al.; We report the DNA sequence of a 9.6-kb region of the Agrobacterium tumefaciens chromosome containing a putative 8-kb chemotaxis operon . The putative operon begins with orf1, whose predicted protein product shows strong sequence identity to methyl-accepting chemotaxis proteins (MCPs), followed by orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, orf10 . All of the identified homologues show a high degree of sequence conservation with their counterparts in the che operons from Sinorhizobium meliloti and Rhodobacter sphaeroides, and are arranged in a similar order . Mutations in orf1 and cheA result in impaired chemotaxis, whereas deletion of orf10, appears to have no effect on chemotaxis or motility . Although the putative operon does not contain a cheW homologue, heterologous probing and PCR using consensus primers indicates that cheW maps elsewhere in the Agrobacterium genome.

J Bacteriol, 1998 Oct, 180(20), 5398 - 405
Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens; Zhu J et al.; The TraR and TraI proteins of Agrobacterium tumefaciens mediate cell-density-dependent expression of the Ti plasmid tra regulon . TraI synthesizes the autoinducer pheromone N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), while TraR is an 3-oxo-C8-HSL-responsive transcriptional activator . We have compared the abilities of 3-oxo-C8-HSL and 32 related compounds to activate expression of a TraR-regulated promoter . In a strain that expresses wild-type levels of TraR, only 3-oxo-C8-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive . Furthermore, many of these compounds were potent antagonists . In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist . We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action . Wild-type A . tumefaciens released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain . However, under all conditions tested, 3-oxo-C8-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with tra gene induction . We conclude that (i) in wild-type strains, only 3-oxo-C8-HSL significantly stimulates tra gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers . When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.

Appl Environ Microbiol, 1998 Oct, 64(10), 3977 - 82
Construction of a range of derivatives of the biological control strain agrobacterium rhizogenes K84: a study of factors involved in biological control of crown gall disease
McClure NC, Ahmadi AR, Clare BG.
The biological control strain Agrobacterium rhizogenes K84 is an effective agent in the control of Agrobacterium pathogens, the causative agents of crown gall disease . A number of factors are thought to play a role in the control process, including production of the specific agrocins 84 and 434, which differ in the spectra of pathogenic strains that they inhibit in vitro . A range of derivatives of strain K84 has been developed with every combination of the three resident plasmids, pAgK84, pAgK434, and pAtK84b, including a plasmid-free strain . These derivatives produced either both, one, or neither of the characterized agrocins 84 and 434 and were isolated by plasmid curing, conjugation, and Tn5 transposon mutagenesis . The ability of the derivative strains to inhibit gall formation on almond roots was compared to that of the wild-type K84 parent . Treatment with the plasmid-free derivative did not result in a significant level of control of an A . rhizogenes pathogen based on numbers or dry weight of galls formed on injured almond roots . The presence of plasmid pAgK84, pAgK434, or pAtK84b significantly enhanced the biological control efficacy of K84 derivatives, and the highest level of control was observed with strains harboring two or more plasmids . The results observed with strains deficient in agrocin 434 production suggest that this product may play an important role in the biological control of A . rhizogenes pathogens . The involvement of plasmid pAgK84b in biological control has not previously been reported . This study supports the conclusion that multiple factors are involved in the success of strain K84 as a biological control agent.

Plant J, 1998 Aug, 15(3), 423 - 33
Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity; Lemcke K et al.; This study tested the morphogenetic potential of 15 open reading frames of the TL-DNA of Agrobacterium rhizogenes strain HRI . These open reading frames were expressed individually under the control of the 35S RNA promoter in transgenic tobacco plants (Nicotiana tabacum L.) . Expression of three T-DNA loci, ORF3n, ORF8 and ORF13, alters plant morphogenesis or the response of transgenic tissues to plant hormones . ORF3n transgenic plants are characterized by retarded flowering, altered internode elongation, altered leaf shape and, in particular, leaf tip necrosis . ORF3n and ORF8 expression reduces the sensitivity to auxin and cytokinin in combination or auxin alone . Tetracycline-dependent expression of ORF13 overcomes a selection of low levels of expression during plant regeneration and reveals a strong inhibitory effect of the ORF13 gene product on cell division and cell elongation . We conclude that the A . rhizogenes TL-DNA harbors genetic information that is important for pathogenicity apart from the well studied rol genes . We propose that these genes play mainly a negative regulatory role during pathogenesis . Moreover, these loci might be relevant to successful infections in specific host plants.

Mol Cells, 1998 Aug 31, 8(4), 393 - 400
Molecular characterization of the virulence gene virG of pTiKU 12; Lee SH et al.; The virulence (vir) genes of Agrobacterium tumefaciens KU12, a Korean strain, were not induced by acetosyringone and the strain showed weak tumor forming ability and broad plant host ranges . We identified complete nucleotide sequence of virG of pTiKU12, an octopine Ti plasmid of this strain . When it was compared with those of other Ti plasmids, pTiKU12 virG contained an open reading frame (ORF) of 726 nucleotides which showed much lower homology (about 77%) than those (above 98%) already known among octopine Ti plasmids and it started with GTG codon instead of TTG found in other Ti plasmids . Only two vir boxes and one promoter region were confirmed in 5'-untranslated region instead of three vir boxes and two promoters which were found in pTiA6 virG . Nevertheless, important amino acids for the functional activity of VirG were so conserved that the virG included in pUCDG could complement a virG mutant Agrobacterium tumefaciens Mx19 in beta-galactosidase activity assays and on plant tumor tests.

Plant Mol Biol, 1998 Oct, 38(3), 393 - 406
Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre; Vergunst AC et al.; The Cre/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana . Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene . To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector had to circularize at the T-DNA left- and right-border sequences prior to site-specific integration) or two lox sequences (this vector allowed circularization at the lox sequences within the T-DNA either prior to or after random integration, followed by targeting of the circularized vector), respectively . Furthermore, to control the reversibility of the integration reaction, Cre recombinase was provided transiently by using a cotransformation approach . One precise stable integrant was found amongst the recombinant calli obtained after transformation with a double-lox T-DNA vector . The results indicate that Agrobacterium-mediated transformation can be used as a tool to obtain site-specific integration.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1994 Nov, 27(4), 206 - 10
Light-emitting aeromonas and plesiomonas generated by transconjugating luxAB from Escherichia coli; Huang MZ et al.; The transposon derivative has been placed on a transposition suicide vector to yield pDB30 in Escherichia coli WA803 . A simple method, using a Tn5 derivative Tn5-Lux, has been successfully devised for the introduction and stable expression of the bioluminescence property in Pseudomonas sp., Agrobacterium sp., and Rhizobium sp . In this study, there was also successful mating between Escherichia coli WA803(pDB30) and strains of Acromonas hydrophila and Plesiomonas shigelloides . These bacteria emitted bioluminescence after they gained pDB30 by transconjugation.

Nat Biotechnol, 1998 Sep, 16(9), 839 - 42
Agrobacterium tumefaciens-mediated transformation of filamentous fungi; de Groot MJ et al.; Agrobacterium tumefaciens transfers part of its Ti plasmid, the T-DNA, to plant cells during tumorigenesis . It is routinely used for the genetic modification of a wide range of plant species . We report that A . tumefaciens can also transfer its T-DNA efficiently to the filamentous fungus Aspergillus awamori, demonstrating DNA transfer between a prokaryote and a filamentous fungus . We transformed both protoplasts and conidia with frequencies that were improved up to 600-fold as compared with conventional techniques for transformation of A . awamori protoplasts . The majority of the A . awamori transformants contained a single T-DNA copy randomly integrated at a chromosomal locus . The T-DNA integrated into the A . awamori genome in a manner similar to that described for plants . We also transformed a variety of other filamentous fungi, including Aspergillus niger, Fusarium venenatum, Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa, and the mushroom Agaricus bisporus, demonstrating that transformation using A . tumefaciens is generally applicable to filamentous fungi.

Nahrung, 1998 Aug, 42(3-4), 128 - 30
Production of genetically modified lysozymes having extreme heat stability and antimicrobial activity against gram negative bacteria in yeast and in plant; Kato A et al.; Hen egg white lysozyme was genetically modified to have extreme heat stability and strong antimicrobial activity against Gram negative bacteria and the modified lysozymes were secreted in yeast and tobacco . Complementary DNA encoding lysozyme was subjected to site-directed mutagenesis to have the Asn-X-Thr(Ser) sequence that is the signal for asparagine-linked glycosylation at the positions 49 . The glycosyl lysozyme enhanced heat stability was expressed in the yeast carrying the modified lysozyme cDNA . The expression amount of glycosyl lysozyme was about 10 mg/l of yeast culture medium . Using the same yeast expression system, the lysozyme enhanced antimicrobial action by inserting hydrophobic penta-peptide at the C-terminus were secreted in a small amount (less than 100 micrograms/l in the yeast culture medium) . These cDNA constructs of modified lysozymes were engineered into tabacco through Agrobacterium-mediated transformation in order to construct antimicrobial plant . The expression of lysozymes was confirmed by the reverse transcriptional PCR, SDS-PAGE analysis and lytic activity of transformants of tobacco . The transformant having the highest lytic activity expressed about 40 micrograms of lysozyme per g of leaf tissue.

Nahrung, 1998 Aug, 42(3-4), 125 - 7
Genetic engineering for high methionine grain legumes; Muntz K et al.; Methionine (Met) is the primary limiting essential amino acid in grain legumes . The imbalance in amino acid composition restricts their biological value (BV) to 55 to 75% of that of animal protein . So far improvement of the BV could not be achieved by conventional breeding . Therefore, genetic engineering was employed by several laboratories to resolve the problem . Three strategies have been followed . A) Engineering for increased free Met levels; B) engineering of endogenous storage proteins with increased numbers of Met residues; C) transfer of foreign genes encoding Met-rich proteins, e.g . the Brazil nut 2S albumin (BNA) and its homologue from sunflower, into grain legumes . The latter strategy turned out to be most promising . In all cases the gene was put under the control of a developmentally regulated seed specific promoter and transferred into grain legumes using the bacterial Agrobacterium tumefaciens-system . Integration into and copy numbers in the plant genome as well as Mendelian inheritance and gene dosage effects were verified . After correct precursor processing the mature 2S albumin was intracellularly deposited in protein bodies which are part of the vacuolar compartment . The foreign protein amounted to 5 to 10% of the total seed protein in the best transgenic lines of narbon bean (Vicia narbonensis L., used in the authors' laboratories), lupins (Lupinus angustifolius L., used in CSIRO, Australia), and soybean (Glycine max (L.) Merr., used by Pioneer Hi-Bred, Inc., USA) . In the narbon bean the increase of Met was directly related to the amount of 2S albumin in the transgenic seeds, but in soybean it remained below the theoretically expected value . Nevertheless, trangenic soybean reached 100%, whereas narbon bean and lupins reached approximately 80% of the FAO-standard for nutritionally balanced food proteins . These results document that the Met problem of grain legumes can be resolved by genetic engineering.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 759 - 68
Evaluation of the relatedness of Brucella spp . and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp . nov., a new species with a closer relationship to Brucella spp; Velasco J et al.; The relatedness of Brucella spp . and Ochrobactrum anthropi was studied by protein profiling, Western blot, immunoelectrophoresis and 16S rRNA analysis . Whole-cell and soluble proteins of brucellae and O . anthropi showed serological cross-reactivities quantitatively and qualitatively more intense than those existing with similar extracts of Agrobacterium spp . Numerical analysis of Western blot profiles of whole-cell extracts showed that O . anthropi LMG 3301 was closer to Brucella spp . than to O . anthropi LMG 3331T, a result not obtained by protein profiling . These differences were not observed by Western blot with soluble fractions, and immunoelectrophoretic analyses suggested that this was due to destruction of conformational epitopes in Western blot procedures with the subsequent simplification of antigenic profile . Analysis of the 16S rRNA sequences of strains previously used in the species definition confirmed that strain LMG 3301, and also LMG 3306, were closer to the brucellae, and that LMG 3331T was in a separate cluster . The LMG 3301 and the LMG 3331T clusters could also be separated by their different colistin sensitivity and by PCR with 16S rRNA Brucella primers, and both methods showed strains of both clusters among clinical isolates classified as O . anthropi by conventional tests . These results and those of previous DNA-DNA hybridization studies {Holmes, B., Popoff, M., Kiredjian, M . & Kersters, K . (1988) . Int J Syst Bacteriol 38, 406-416} show that the LMG 3301 cluster and related clinical isolates should be given a new species status for which the name Ochrobactrum intermedium sp . nov . is proposed (type strain is LMG 3301T=NCTC 12171T = CNS 2-75T).

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 687 - 99
Rhizobium huautlense sp . nov., a symbiont of Sesbania herbacea that has a close phylogenetic relationship with Rhizobium galegae; Wang ET et al.; The nitrogen-fixing rhizobial symbionts of Sesbania herbacea growing in the nature reserve at the Sierra de Huautla, Mexico, were isolated and characterized . All 104 isolates together with the type strain for Rhizobium galegae, HAMBI 540T, had similar 16S rRNA genes as revealed by PCR-RFLP analysis . Similarity in the sequences of the 16S rRNA genes placed the isolates on a phylogenetic branch shared with R . galegae . Among 66 randomly selected isolates, three closely related electrophoretic alloenzyme types (ETs) were identified, which were distinct from 10 ETs distinguished among 23 strains of R . galegae . A new species Rhizobium huautlense, represented by the Sesbania isolate SO2T, is proposed based upon low estimates of DNA relatedness between our chosen type strain and the type strains for the other species, the dissimilarity of the nucleotide sequence of the 16S rRNA genes, and their distinct ETs compared with R . galegae . The description of R . huautlense is significant because in the reconstruction of the phylogeny at R . huautlense there was a shift in the node of the branch of Agrobacterium vitis relative to that of R . galegae . The revised phylogenetic tree would tend to indicate common ancestry between R . galegae and Rhizobium leguminosarum.

Plant Physiol, 1998 Sep, 118(1), 51 - 8
Expression of the yeast FRE genes in transgenic tobacco; Samuelsen AI et al.; Two yeast genes, FRE1 and FRE2 (encoding Fe(III) reductases) were placed under the control of the cauliflower mosaic virus 35S promoter and introduced into tobacco (Nicotiana tabacum L.) via Agrobacterium tumefaciens-mediated transformation . Homozygous lines containing FRE1, FRE2, or FRE1 plus FRE2 were generated . Northern-blot analyses revealed mRNA of two different sizes in FRE1 lines, whereas all FRE2 lines had mRNA only of the expected length . Fe(III) reduction, chlorophyll contents, and Fe levels were determined in transgenic and control plants under Fe-sufficient and Fe-deficient conditions . In a normal growth environment, the highest root Fe(III) reduction, 4-fold higher than in controls, occurred in the double transformant (FRE1 + FRE2) . Elevated Fe(III) reduction was also observed in all FRE2 and some FRE1 lines . The increased Fe(III) reduction occurred along the entire length of the roots and on shoot sections . FRE2 and double transformants were more tolerant to Fe deficiency in hydroponic culture, as shown by higher chlorophyll and Fe concentrations in younger leaves, whereas FRE1 transformants did not differ from the controls . Overall, the beneficial effects of FRE2 were consistent, suggesting that FRE2 may be used to improve Fe efficiency in crop plants.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 349 - 56
Phylogeny of Rhizobium galegae with respect to other rhizobia and agrobacteria; Terefework Z et al.; PCR-RFLP with nine restriction enzymes was applied to the 16S and 23S rRNA genes of 42 rhizobial and agrobacterial strains to determine the phylogenetic position of Rhizobium galegae and increase the understanding of the evolution of ribosomal operons . The strains were selected based on previous phylogenetic studies . PCR primers were designed so that they amplified a 2.3 kb fragment of the 23S rRNA gene (excluding the B8 loop) . Universal primers rD1 and fD1 were used to amplify the full-length 16S rRNA . The RFLP analysis resulted in 27 and 32 different restriction patterns for 16S and 23S, respectively . The RFLP patterns were transformed to genetic distances and dendrograms were constructed from the data using the unweighted pair group method with averages . The shapes of the dendrograms derived from the analysis of the 16S and 23S rRNA genes correlated well, with only a few strains having different positions . The 23S tree generally had deeper branching than the 16S tree, allowing better discrimination between species and strains . The combined data from the two analyses described 36 genotypes . The eight R . galegae strains formed a homogeneous cluster in all dendrograms . The RFLP analysis was confirmed by partial sequence analysis of the 16S rRNA gene (the first 800 bp), which correlated well with full-length 16S rRNA sequence analysis . The 16S data placed R . galegae near the genus Agrobacterium with Agrobacterium vitis as its nearest neighbour, whereas in the 23S and the combined dendrograms it showed closer affinity to the genus Rhizobium.

Appl Environ Microbiol, 1998 Sep, 64(9), 3368 - 75
Biodegradation of atrazine by Agrobacterium radiobacter J14a and use of this strain in bioremediation of contaminated soil; Struthers JK et al.; We examined the ability of a soil bacterium, Agrobacterium radiobacter J14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites . J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 microgram of {14C-U-ring}atrazine ml-1 in 72 h with a concurrent increase in the population size from 7.9 x 10(5) to 5.0 x 10(7) cells ml-1 . Under these conditions cells mineralized the {ethyl-14C}atrazine and incorporated approximately 30% of the 14C into the J14a biomass . Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase . Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine . The addition of 10(5) J14a cells g-1 into soil with a low indigenous population of atrazine degraders treated with 50 and 200 microgram of atrazine g-1 soil resulted in two to five times higher mineralization than in the noninoculated soil . Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times . However, J14a introduction (10(5) cells g-1) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization.

Mol Plant Microbe Interact, 1998 Sep, 11(9), 855 - 9
Modified development in transgenic tobacco plants expressing a rolA::GUS translational fusion and subcellular localization of the fusion protein; Vilaine F et al.; The rolA gene is transferred naturally by Agrobacterium rhizogenes to the genome of host plants, where it induces dramatic changes in development of transformed plants, including dwarfism and leaf wrinkling . The predicted translation product of the rolA gene is a small (11.4 kDa), basic (pI = 11.2) protein, which has no clearly significant similarity to sequences in the data bases . We have introduced into the tobacco genome a gene encoding a rolA::GUS fusion protein . Expression of this gene led to synthesis of an RNA and a protein of expected size, and the transformed plants exhibited the dwarfism and leaf wrinkling typical of rolA plants, but to a lesser degree than plants transformed with the wild-type rolA gene . The distribution of beta-glucuronidase (GUS) activity was compared in subcellular fractions of leaf extracts from plants expressing either the rolA::gus gene or a control gus construct . As expected, in the control plants, GUS activity was essentially cytosolic . In contrast, in plants expressing the rolA::gus gene the highest specific activity was associated with the plasmalemma fraction.

J Bacteriol, 1998 Sep, 180(17), 4392 - 400
Molecular cloning and characterization of cgs, the Brucella abortus cyclic beta(1-2) glucan synthetase gene: genetic complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB mutants; Inon de Iannino N et al.; The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant . The complemented strains recovered the synthesis of cyclic beta(1-2) glucan, motility, virulence in A . tumefaciens, and nitrogen fixation in R . meliloti; all traits were strictly associated with the presence of an active cyclic beta(1-2) glucan synthetase protein in the membranes . Nucleotide sequencing revealed the presence in B . abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R . meliloti NdvB . Four regions of the B . abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R . meliloti NdvB . Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic beta(1-2) glucan synthesis . Confirmation of the identity of this protein as B . abortus cyclic beta(1-2) glucan synthetase was done by the construction of a B . abortus Tn3-HoHo1 insertion mutant that does not form cyclic beta(1-2) glucan and lacks the 316.2-kDa membrane protein . The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic beta(1-2) glucan may be a virulence factor in Brucella infection.

Arch Biochem Biophys, 1998 Sep 1, 357(1), 13 - 21
Cloning and expression of the glgC gene from Agrobacterium tumefaciens: purification and characterization of the ADPglucose synthetase; Uttaro AD et al.; The gene encoding ADPglucose synthetase (EC 2.7.7.27) from Agrobacterium tumefaciens was isolated and expressed in Escherichia coli . The recombinant protein was purified to electrophoretic homogeneity in steps including ion-exchange and hydrophobic chromatography . The same purification procedure was utilized to purify ADPglucose synthetase from A . tumefaciens cells . The enzymes from the two sources were purified and characterized and were found to have identical kinetic, regulatory, and structural properties . In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only one polypeptide band of 50 kDa was detected . In immunoblotting following electrophoresis, the 50-kDa band reacted with antibodies raised against the Escherichia coli ADPglucose synthetase; there was no reaction with antibodies raised against the spinach enzyme . The immunoreactivity of the A . tumefaciens ADPglucose synthetase was confirmed in antibody neutralization assays . Using gel filtration, the native enzyme was shown to be a tetramer . Fructose 6-phosphate and pyruvate were the most effective activators of the enzyme; maximal activation was observed in the ADPglucose synthesis direction, in which the enzyme was activated about ninefold by fructose 6-phosphate and fivefold by pyruvate . Both activators increased the affinity of the enzyme for the substrates ATP and glucose 1-phosphate . Inorganic orthophospate, ADP, AMP, and pyridoxal phosphate behaved as inhibitors of the enzyme . The distinctive regulatory properties of the enzyme from A . tumefaciens are compared with those of two enterobacterial enzymes and discussed in the context of their deduced amino acid sequences .

Plant Cell, 1998 Aug, 10(8), 1277 - 94
Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development; Uwer U et al.; Embryo formation is the first patterning process during vegetative plant growth . Using transposons as insertional mutagens in Arabidopsis, we identified the mutant edd1 that shows embryo-defective development . The insertion mutation is lethal, arresting embryo growth between the globular and heart stages of embryonic development . The mutant phenotype cosegregates with a transposed Dissociation element . Sequences flanking the transposed element were isolated and used to isolate a full-length cDNA clone representing the wild-type EDD1 gene . Complementation of the mutant through Agrobacterium-mediated gene transfer of an EDD1 wild-type copy as well as loss of the transposon concomitant with phenotypic reversion demonstrated that the transposon had caused the mutation . Based on homology to Escherichia coli, the EDD1 gene is predicted to encode a novel glycyl-tRNA synthetase (GlyRS) that has not been identified previously in higher plants . An N-terminal portion of the plant protein is able to direct a marker protein into pea chloroplasts . Thus, the gene identified by the embryo-defective insertion mutation encodes a GlyRS homolog, probably acting within the plastidic compartment.

Mol Microbiol, 1998 Jul, 29(1), 165 - 77
Sequence variations in alleles of the avirulence gene avrPphE.R2 from Pseudomonas syringae pv . phaseolicola lead to loss of recognition of the AvrPphE protein within bean cells and a gain in cultivar-specific virulence; Stevens C et al.; The bean halo blight pathogen, Pseudomonas syringae pv . phaseolicola (Psph), is differentiated into nine races based on the presence or absence of five avirulence (avr) genes in the bacterium, which interact with corresponding resistance genes . R1-R5, in Phaseolus vulgaris . The resistance gene R2 is matched by avrPphE, which is located adjacent to the cluster of hrp genes that are required for pathogenicity of Psph . Although only races 2, 4, 5 and 7 are avirulent on cultivars with R2 (inducing the hypersensitive response; HR), homologues of avrPphE are present in all races of Psph . DNA sequencing of avrPphE alleles from races of Psph has demonstrated two routes to virulence: via single basepair changes conferring amino acid substitutions in races 1, 3, 6 and 9 and an insertion of 104bp in the allele in race 8 . We have demonstrated that these base changes are responsible for the difference between virulence and avirulence by generating transconjugants of a virulent race harbouring plasmids expressing the various alleles of avrPphE . Agrobacterium tumefaciens-directed expression of avrPphE from race 4 in bean leaves induced the HR in a resistance gene-specific manner, suggesting that the AvrPphE protein is alone required for HR induction and is recognized within the plant cell . The allele from race 6, which is inactive if expressed in Psph, elicited a weak HR if expressed in planta, whereas the allele from race 1 did not . Our results suggest that the affinity of interaction between AvrPphE homologues and an unknown plant receptor mediates the severity of the plant's response . Mutation of avrPphE alleles did not affect the ability to colonize bean from a low level of inoculum . The avirulence gene avrPphB, which matches the R3 resistance gene, also caused a gene-specific HR following expression in the plant after delivery by A . tumefaciens.

Mol Microbiol, 1998 Jul, 29(1), 125 - 38
A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence; Sola-Landa A et al.; Two mutants showing increased sensitivity to polycations and surfactants were obtained by transposon mutagenesis of virulent Brucella abortus 2308 Nalr . These mutants showed no obvious in vitro growth defects and produced smooth-type lipopolysaccharides . However, they hardly multiplied or persisted in mouse spleens, displayed reduced invasiveness in macrophages and HeLa cells, lost the ability to inhibit lysosome fusion and were unable to replicate intracellularly . Subsequent DNA analyses identified a two-component regulatory system {Brucella virulence related (Bvr)} with a regulatory (BvrR) and sensory (BvrS) protein . Cloning of bvrR in the BvrR-deficient mutant restored the resistance to polycations and, in part, the invasiveness and the ability to multiply intracellularly . BvrR and BvrS were highly similar (87-89% and 70-80% respectively) to the regulatory and sensory proteins of the chromosomally encoded Rhizobium meliloti Chvl-ExoS and Agrobacterium tumefaciens Chvl-ChvG systems previously shown to be critical for endosymbiosis and pathogenicity in plants . Divergence among the three sensory proteins was located mostly within a periplasmic domain probably involved in stimulus sensing . As B . abortus, R . meliloti and A . tumefaciens are phylogenetically related, these observations suggest that these systems have a common ancestor that has evolved to sense stimuli in plant and animal microbial environments.

Plant Mol Biol, 1998 Aug, 37(6), 1079 - 84
Construction of a new vector conferring methotrexate resistance in Nicotiana tabacum plants; Irdani T et al.; A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation . Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18 . Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx . Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations . Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants . The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C . albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding.

J Bacteriol, 1998 Aug, 180(16), 4300 - 2
Agrobacterium VirE2 proteins can form a complex with T strands in the plant cytoplasm; Gelvin SB; Wild-type VirE2 and VirD2 proteins from Agrobacterium tumefaciens contain nuclear targeting sequences (NLS) that are likely involved in directing transferred T strands to the plant nucleus . An A . tumefaciens virE2 virD2DeltaNLS double mutant was able to form tumors on VirE2-producing transgenic tobacco but not on wild-type tobacco . Because this mutant bacterial strain contains no known T-strand nuclear targeting signal, the data indicate that wild-type VirE2 proteins produced by the plant can interact with the T strands in the plant cytoplasm and direct them to the nucleus.

Biotechnol Appl Biochem, 1998 Aug, 28 ( Pt 1), 33 - 8
Peroxidase production in vitro by Armoracia lapathifolia (horseradish)-transformed root cultures: effect of elicitation on level and profile of isoenzymes; Flocco CG et al.; Transformed roots of Armoracia lapathifolia (horseradish) were established by infection with Agrobacterium rhizogenes LBA 9402 . They were used as a culture system in vitro for peroxidase production in vitro, to avoid many of the problems that affect the traditional production from field-grown species of Armoracia sp . The time course of growth of these cultures showed that total peroxidase attained maximum levels at the end of the exponential growth phase . At this stage of culture, elicitation assays were performed with AgNO3 and CuSO4 as abiotic elicitors and with fungal extracts of Verticillum sp., Monodyctis cataneae and Aspergillus niger as biotic elicitors . The best results were obtained with Verticillum sp., 24 h after elicitation, with an increase of approx . 100% in peroxidase activity . The isoenzyme pattern analysed by isoelectric focusing revealed predominantly basic and acidic isoenzymes in both plant roots and transformed root cultures . Elicited samples showed a similar isoenzyme pattern with a slight increase in basic isoenzymes.

Proc Natl Acad Sci U S A, 1998 Aug 4, 95(16), 9105 - 10
Interaction of the DNA modifying proteins VirD1 and VirD2 of Agrobacterium tumefaciens: analysis by subcellular localization in mammalian cells; Relic B et al.; Interaction between Agrobacterium tumefaciens and plants provides a unique example of interkingdom gene transfer . Agrobacterium, a plant pathogen, is capable to stably transform the plant cell with a segment of its own DNA called T-DNA (transferred DNA) . This process depends, among others, on the specialized bacterial virulence proteins VirD1 and VirD2 that excise the T-DNA from its adjacent sequences . Subsequent to transfer to the plant cell, the virulence protein VirD2, through its nuclear localization signal (NLS), is believed to guide the T-DNA to the nucleus . The T-DNA then is integrated into the plant genome . Although both of these proteins are essential for bacterial virulence, physical interaction of them has not been analyzed so far . We studied associations between these proteins by expressing them in mammalian cells and by testing for intracellular localization and colocalization . When expressed in human cells {HeLa, human embryo kidney (HEK) 293}, the VirD2 protein homogeneously distributed over the nucleoplasm . The presence of any of two NLSs, on the N and C termini of VirD2, was sufficient for its efficient nuclear localization whereas deletion of both NLSs rendered the protein cytoplasmic . However, this double NLS mutant was translocated to the nucleus in the presence of wild-type VirD2 protein, implying VirD2-VirD2 interaction . The VirD1 protein, by itself localized in the cytoplasm, moved to the nucleus when coexpressed with the VirD2 protein, suggesting VirD1-VirD2 interaction . This interaction was confirmed by coimmunoprecipitation tests . Of interest, both proteins coimported to the nucleus showed a similar, peculiar sublocalization . The data are discussed in terms of functions of the VirD proteins . In addition, coimport of proteins into nuclei is suggested as a useful system in studying individual protein-protein interactions.

J Bacteriol, 1998 Aug, 180(15), 3933 - 9
The conjugal intermediate of plasmid RSF1010 inhibits Agrobacterium tumefaciens virulence and VirB-dependent export of VirE2; Stahl LE et al.; Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation . The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation . The movement of the transferred DNA and VirE2 from A . tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins . Further, the movement of the IncQ broad-host-range plasmid RSF1010 between Agrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system . Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants . Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.

Plant J, 1998 Mar, 13(5), 707 - 16
Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension; Mathur J et al.; A protocol for establishment and high-frequency Agrobacterium-mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags . Thirty-two self-circularized T-DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR) . By bidirectional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J-domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen-specific transcript . In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.

DNA Res, 1998 Apr 30, 5(2), 87 - 93
Structural characterization of the virB operon on the hairy-root-inducing plasmid A4; Liang Y et al.; The hairy-root-inducing plasmid A4 (pRiA4) is capable of conferring tumorigenic symptoms on plants upon infection by its host bacterium, Agrobacterium rhizogenes . The virB operon on pRiA4 has been sequenced and found to be composed of 11 genes, virB1 to virB11, whose products mostly appear to be associated with the cell membrane . A novel structural characteristic is frequent overlappings between the translation termination and initiation codons of adjacent genes . This is indicative of fine tuning of relative translation frequencies for each VirB protein . These results support the view that VirB multisubunit complexes provide facilities for T-DNA transfer at the bacterial cell membrane . The structural organization of the pRiA4 virB operon was essentially identical to that of the previously reported virB operons of tumor-inducing plasmids, pTiC58 and pTiA6, and the corresponding VirB proteins of the three plasmids were extremely homologous to one another . On the basis of the structural similarity of each VirB protein, the phylogenetic relationship among pRiA4, pTiC58, and pTiA6 is discussed.

Biotechnology (N Y), 1995 Oct, 13(10), 1090 - 3
High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds; Fiedler U et al.; We have used transgenic tobacco seeds to produce large amounts of a functionally active engineered antibody . A gene infusion encoding an antigen-binding single chain Fv protein (scFv) that recognizes the hapten oxazolone was constructed and used as a model . After characterization in a bacterial expression system ,the scFv gene was cloned into a plant expression cassette conferring seed specific expression, and transferred using Agrobacterium-mediated transformation, into Nicotiana tabacum . The expressed scFv could be detected in the developing as well as ripe seeds of regenerated transgenic plants, and the functionally active scFv is stabaly deposited and accumulates up to 0.67% of the total soluble seed protein . After storage of ripe transgenic tobacco seeds for one year at room temperature there was no loss of scFv protein or its antigen-binding activity.

Plant Mol Biol, 1998 Jul, 37(5), 829 - 38
Expression of the Brazil nut methionine-rich protein and mutants with increased methionine in transgenic potato; Tu HM et al.; A cDNA encoding the methionine-rich (19 mol% Met) protein in Brazil nut was placed under the regulation of CaMV 35S promoter and nopaline synthase terminator and introduced into the potato cultivar Russet Burbank via Agrobacterium-mediated transformation . To further enhance the Met content in the transgenic plants, chimeric genes containing four mutant constructs, BoxIa (with 5 additional Met), BoxIIa (2 additional Met), BoxIaIIa (7 additional Met), and BoxIIa2 (7 additional Met), were also generated by sequence modifications of the cDNA and transferred into potato . Analysis of the microtubers and leaves of the transgenic potato plants revealed, in general, with the exception of the BoxIIa2, the presence of mRNA transcripts of the expected size and the correctly processed Met-rich 9 kDa subunit polypeptides . The expression levels in the leaves among the various constructs and individual transgenic plants varied between <0.01% and 0.2% of total protein . The corresponding expression in the tubers was usually 2- to 4-fold lower than in leaves . In the case of BoxIIa2, which contains two tandem repeats of the BoxIIa mutant sequence, a larger (10.5-11 kDa) polypeptide was detected . These findings demonstrated that it is feasible to exploit the variable region of the Brazil Nut 2S protein for enhanced Met contents and perhaps for other desirable properties.

Ann N Y Acad Sci, 1998 Jun 30, 851, 147 - 51
Regulation of heat-shock response in bacteria; Segal G et al.; Stress response in bacteria is essential for effective adaptation to changes in the environment, as well as to the changes in the physiological state of the bacterial culture itself . This response is mediated by global regulatory mechanisms affecting several pathways . It now appears that these regulatory mechanisms operate by transcriptional control, translational control, and proteolysis . One example to be discussed extensively is the heat-shock response . In Escherichia coli, where it has been studied initially and most extensively, the expression of the heat-shock operon is transcriptionally controlled by the employment of the heat-shock transcription factor sigma 32, that recognizes specific heat-shock promoters . Later studies indicated that in most bacteria the control of the major heat-shock genes is much more complicated, and involves additional--or alternative--control channels . These regulatory elements will be reviewed looking at the groE and dnaK operons . These operons, coding for the bacterial equivalent of Hsp10+60 and Hsp70, respectively, contain in many bacteria a conserved regulatory inverted repeat (IR = CIRCE), and are transcribed either by the vegetative sigma factor--sigma 70--or by a sigma 32-like factor . The IR functions at the DNA level as a repressor binding site and also controls the half life of the transcript . In addition, in Agrobacterium tumefaciens there also exists a system for mRNA processing that involves a temperature-controlled cleavage of the groE transcript.

Plant Physiol, 1998 Jul, 117(3), 841 - 9
The never ripe mutant provides evidence that tumor-induced ethylene controls the morphogenesis of agrobacterium tumefaciens-induced crown galls on tomato stems
Aloni R, Wolf A, Feigenbaum P, Avni A, Klee HJ.
We confirm the hypothesis that Agrobacterium tumefaciens-induced galls produce ethylene that controls vessel differentiation in the host stem of tomato (Lycopersicon esculentum Mill.) . Using an ethylene-insensitive mutant, Never ripe (Nr), and its isogenic wild-type parent we show that infection by A . tumefaciens results in high rates of ethylene evolution from the developing crown galls . Ethylene evolution from isolated internodes carrying galls was up to 50-fold greater than from isolated internodes of control plants when measured 21 and 28 d after infection . Tumor-induced ethylene substantially decreased vessel diameter in the host tissues beside the tumor in wild-type stems but had a very limited effect in the Nr stems . Ethylene promoted the typical unorganized callus shape of the gall, which maximized the tumor surface in wild-type stems, whereas the galls on the Nr stems had a smooth surface . The combination of decreased vessel diameter in the host and increased tumor surface ensured water-supply priority to the growing gall over the host shoot . These results indicate that in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown-gall morphogenesis.

Plant Physiol, 1998 Jul, 117(3), 809 - 20
Induction of defense-related responses in Cf9 tomato cells by the AVR9 elicitor peptide of cladosporium fulvum is developmentally regulated
Honee G, Buitink J, Jabs T, De Kloe J, Sijbolts F, Apotheker M, Weide R, Sijen T, Stuiver M, De Wit PJ.
The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene . To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated . Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes . Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation . Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained . Transgenic F1 seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks . However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene . It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells . These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.

Mol Microbiol, 1998 Jun, 28(5), 1027 - 38
Natural competence for DNA transformation in Helicobacter pylori: identification and genetic characterization of the comB locus; Hofreuter D et al.; The gram-negative bacterial pathogen Helicobacter pylori, an important aetiological agent of gastroduodenal disease in humans, belongs to a group of bacterial species displaying competence for genetic transformation . Here, we describe the comB gene locus of H . pylori involved in DNA transformation competence . It consists of a cluster of four tandemly arranged genes with partially overlapping open reading frames, orf2, comB1, comB2 and comB3, constituting a single transcriptional unit . Orf2 encodes a 37-amino-acid peptide carrying a signal sequence, whereas comB1, comB2 and comB3 produce 29 kDa, 38 kDa and 42 kDa proteins, respectively, as demonstrated by immunoblotting with specific antisera . For Orf2 and ComB1, no homologous proteins were identified in the database . For ComB3, the best homologies were found with TraS/TraB from the Pseudomonas aeruginosa conjugative plasmid RP1 and TrbI of plasmid RP4, VirB10 from the Ti plasmid of Agrobacterium tumefaciens and PtlG, a protein involved in secretion of pertussis toxin of Bordetella pertussis . Defined transposon knock-out mutants in individual comB genes resulted in transformation-defective phenotypes ranging from a 90% reduction to a complete loss of the natural transformation efficiency . The comB2 and comB3 genes show homology to HP0528 and HP0527, respectively, located on the cagII pathogenicity island of H . pylori strain 26695.

Mol Biotechnol, 1998 Apr, 9(2), 175 - 9
Half-embryo cocultivation technique for estimating the susceptibility of pea (Pisum sativum L.) and lentil (Lens culinaris Medik.) cultivars to Agrobacterium tumefaciens; Lurquin PF et al.; Longitudinally sliced embryonic axes from pea and lentil mature seeds cocultivated with A . tumefaciens carrying a gus reporter gene in its T-DNA provided a convenient means to evaluate the efficiency of gene transfer to tissues in different cultivars and cocultivation conditions . Use of this technique demonstrated wide variation in susceptibility to Agrobacterium among several pea and lentil commercial genotypes.

Mol Biotechnol, 1998 Apr, 9(2), 155 - 9
A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation; McCormac AC et al.; The introduction of binary plasmids into Agrobacterium hosts for Agrobacterium-mediated transformation of plants is most readily achieved by electroporation . However, occasionally, no transformed colonies are recovered and the transformation program is delayed . Poor transformation rates are commonly associated with particular combinations of Agrobacterium strains and plasmid-selection markers . In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA . It is also important to optimize the level of antibiotic for the selection of transformed colonies . In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized . We have observed that there is significant variation between transformed Agrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of three Argobacterium tumefaciens (EHA101, LBA4404, C58) and two Agrobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin(res), kanamycin(res), and gentamycin(res)).

Mol Plant Microbe Interact, 1998 Jul, 11(7), 668 - 83
Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration; Mysore KS et al.; VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer . In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence . Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome . In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration . Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border . The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter . Approximately 0.4% of the tobacco cell clusters infected by a wild-type A . tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc) . However, using an omega-mutant A . tumefaciens strain harboring the same binary vector, we did not detect any blue staining . Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A . tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution . Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration . To determine whether the use of the T-DNA right border is altered in those few tumors generated by A . tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors . Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A . tumefaciens was wild-type or contained an omega mutation . In addition, a mutant VirD2 protein lacking the omega domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein . Finally, we investigated the role of various amino acids of the omega and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts . Deletion of the omega domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein . Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

J Biotechnol, 1998 Mar 26, 61(1), 1 - 13
Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterization as new member of cyclic amidases; May O et al.; A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity with a yield of 77% using a three-step purification procedure . The active enzyme is a tetramer consisting of four identical subunits, each with a molecular mass of 49670 Da as determined by mass spectrometry . The N-terminal amino acid sequence of the enzyme indicates sequence identities to cyclic amidases involved in the nucleotide metabolism as the D-hydantoinase from Agrobacterium radiobacter (53%), the D-selective dihydropyrimidinase from Bacillus stearothermophilus (38%), the allantoinase from Rana catesbeiana (26%), as well as to the catalytic subunit of the urease from Helicobacter pylori (50%) . However, all studies based on substrate-dependent growth, induction and catalytic behavior documented the novelty of the bacterial hydantoinase and that its physiological role is not related to any of these enzymes or known metabolic pathways . Its substrate specificity differs from hydantoinases listed in Enzyme Nomenclature and is rather more predominant for the cleavage of aryl- than for alkyl-hydantoin derivatives . It is shown that the stereoselectivity of this enzyme depends on the substrate used for bioconversion: although it is strictly L-selective for the cleavage of D,L-5-indolylmethylhydantoin, it appears to be D-selective for the hydrolysis of D,L-methylthioethylhydantoin . Due to these findings we conclude that this novel bacterial hydantoinase should be classified as a new member of the EC-group 3.5.2 of cyclic amidases.

Biosci Biotechnol Biochem, 1998 May, 62(5), 882 - 6
Screening, characterization, and cloning of the gene for N-carbamyl-D-amino acid amidohydrolase from thermotolerant soil bacteria; Ikenaka Y et al.; For the production of D-amino acids, thermotolerant bacteria producing N-carbamyl-D-amino acid amidohydrolase were isolated from soil by enrichment culture at 45 degrees C with N-carbamyl-D-amino acids as the sole nitrogen source . The enzyme activities and substrate specificities of these strains were examined by the resting cells reaction . One of the enzymes, produced by Pseudomonas sp . strain KNK003A, was purified and characterized, and the amino acids of its N-terminal region were sequenced . A DNA fragment containing the gene for a thermostable N-carbamyl-D-amino acid amidohydrolase was then cloned into Escherichia coli . The gene encoded a peptide of 312 amino acids, with a calculated molecular weight of 35,000 . The similarity of the deduced amino acid sequences of this enzyme and a related enzyme from a mesophile, Agrobacterium sp . strain KNK712, was 60% . A database was searched for similar sequences.

Biosci Biotechnol Biochem, 1998 May, 62(5), 875 - 81
Isolation of Agrobacterium sp . strain KNK712 that produces N-carbamyl-D-amino acid amidohydrolase, cloning of the gene for this enzyme, and properties of the enzyme; Nanba H et al.; Agrobacterium sp . strain KNK712, which produced N-carbamyl-D-amino acid amidohydrolase (DCase) was isolated from soil . The bacterium had D-specific hydantoinase activity also . Both enzymes are suitable for use in the production of D-amino acids . The DCase gene from Agrobacterium sp . strain KNK712 was cloned into Escherichia coli . The cloned DNA fragment contained one open reading frame, predicted to encode a peptide of 304 amino acids, with a calculated molecular weight of 34,285 . The DCase gene was overexpressed under the control of the lac promoter, and DCase accounted for 50% of the soluble protein in the cells . The enzyme was purified and some properties were investigated . Both the optimum pH and the pH that gave greatest stability were about pH 7.0 . The optimum temperature was 65 degrees C, and the enzyme was stable at 55 degrees C . The enzyme had strict specificity toward N-carbamyl-D-amino acids, and was inhibited by thiol reagents, Cu2+, Hg2+, Ag+, and ammonia.

Appl Environ Microbiol, 1998 Jul, 64(7), 2341 - 5
Root colonization by Agrobacterium tumefaciens is reduced in cel, attB, attD, and attR mutants; Matthysse AG et al.; Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil . Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 10(3) bacteria/cm of root length at the time of inoculation to more than 10(7) bacteria/cm after 10 days . The numbers of cellulose-minus and nonattaching attB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots . On roots of A . thaliana ecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days . The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days . The attachment of A . tumefaciens to cut A . thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel and att mutants . These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.

Planta, 1998 Jul, 205(3), 414 - 9
Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus; Canel C et al.; Cells of Catharanthus roseus (L.) G . Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs) . The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway . Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway . Two such lines accumulated over 200 mg.L-1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity . Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors . High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures . In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis . We conclude that constitutive over-expression of Str is highly desirable for increased TIA production . However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.

Biotechnology (N Y), 1995 Dec, 13(13), 1484 - 7
Expression of the rabies virus glycoprotein in transgenic tomatoes; McGarvey PB et al.; We have engineered tomato plants (Lycopersicon esculentum Mill var . UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface of the rabies virus . The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus . Plants were transformed by Agrobacterium tumefaciens-mediated transformation of cotyledons and tissue culture on selective media . PCR confirmed the presence of the G-protein gene in plants surviving selection . Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants . The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera . The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells . Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells . In light of our previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines.

Nat Biotechnol, 1996 Jun, 14(6), 745 - 50
High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens; Ishida Y et al.; Transformants of maize inbred A188 were efficiently produced from immature embryos cocultivated with Agrobacterium tumefaciens that carried "super-binary" vectors . Frequencies of transformation (independent transgenic plants/embryos) were between 5% and 30% . Almost all transformants were normal in morphology, and more than 70% were fertile . Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis . Between one and three copies of the transgenes were integrated with little rearrangement, and the boundaries of T-DNA were similar to those in transgenic dicotyledons and rice . F1 hybrids between A188 and five other inbreds were transformed at low frequencies.

Nat Biotechnol, 1996 Jun, 14(6), 736 - 40
Genetic transformation of cassava (Manihot esculenta Crantz); Li HQ et al.; Genetic engineering can be used to complement traditional breeding methods in crop plant improvement . Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems . The prerequisites for genetic engineering are efficient transformation and tissue culture systems that allow selection and regeneration of transgenic plants . Cassava, an integral plant for food security in developing countries, has until now been recalcitrant to transformation approaches . We report here a method for regenerating stably transformed cassava plants after cocultivation with Agrobacterium tumefaciens, which opens cassava for future improvement via biotechnology.

Nat Biotechnol, 1996 May, 14(5), 643 - 6
Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene; Grison R et al.; Constitutive overexpression of a protein involved in plant defense mechanisms to disease is one of the strategies proposed to increase plant tolerance to fungal pathogens . A hybrid endochitinase gene under a constitutive promoter was introduced by Agrobacterium-mediated transformation into a winter-type oilseed rape (Brassica napus var . oleifera) inbred line . Progeny from transformed plants was challenged using three different fungal pathogens (Cylindrosporium concentricum, Phoma lingam, Sclerotinia sclerotiorum) in field trials at two different geographical locations . These plants exhibited an increased tolerance to disease as compared with the nontransgenic parental plants.

Nat Biotechnol, 1996 May, 14(5), 624 - 8
Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions; Perl A et al.; Very short exposures of embryogenic calli of Vitis vinifera cv . Superior Seedless grape plants to diluted cultures of Agrobacterium resulted in plant tissue necrosis and subsequent cell death . Antibiotics used for Agrobacterium elimination or as plant selectable markers were not responsible for this necrotic response . Rather, cell death seemed to be oxygen-dependent and correlated with elevated levels of peroxides . Therefore, we studied the effects on necrosis of various combinations of antioxidants during and after grape-Agrobacterium cocultivation . The combination of polyvinylpolypyrrolidone and dithiothreitol was found to improve plant viability . Tissue necrosis was completely inhibited by these antioxidants while Agrobacterium virulence was not effected . These treatments enabled the recovery of stable transgenic grape plants resistant to hygromycin.

Biochim Biophys Acta, 1998 Jun 11, 1385(1), 78 - 88
Agrobacterium tumefaciens beta-glucosidase is also an effective beta-xylosidase, and has a high transglycosylation activity in the presence of alcohols; Watt DK et al.; Agrobacterium tumefaciens beta-glucosidase, Cbg1 was extensively characterised and found to be a retaining aryl-glucosidase and an aryl-xylosidase . Cbg1s specificity for p-nitrophenyl beta-d-xylopyranoside was 73% that for p-nitrophenyl beta-d-glucopyranoside when measured by the ratio kcat/Km . The enzyme also hydrolysed p-nitrophenyl beta-d-fucopyranoside, and p-nitrophenyl beta-d-galactopyranoside with moderate efficiency . The enzyme released only terminal glucose from p-nitrophenyl beta-cellobioside and had a 20 000-fold preference for its natural substrate coniferin over cellobiose as indicated by the ratio kcat/Km . The enzyme was activated in the presence of 20 mM 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, and 1-octanol . In the case of butanol this activation was investigated and shown to be due to transglycosylation activity with over 80% of p-nitrophenyl beta-d-glucopyranoside being converted to 1-butyl beta-d-glucopyranoside in the presence of Cbg1 and 100 mM 1-butanol .

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7203 - 8
Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots; Kohli A et al.; Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism . In the "preintegration" phase, transforming plasmid molecules (either intact or partial) are spliced together . This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences . Subsequently, integration of transgenic DNA into the host genome is initiated . Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus . The resulting transgenic locus may have plant DNA separating the transgenic sequences . Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism . Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically . Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and/or quantitative efficiency of transformation events is not compromised . Our results cast doubt on the role of Agrobacterium vir genes in the integration process.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7057 - 62
The Ti plasmid increases the efficiency of Agrobacterium tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid; Bohne J et al.; The T-DNA transfer apparatus of Agrobacterium tumefaciens mediates the delivery of the T-DNA into plant cells, the transfer of the IncQ plasmid RSF1010 into plant cells, and the conjugal transfer of RSF1010 between Agrobacteria . We show in this report that the Agrobacterium-to-Agrobacterium conjugal transfer efficiencies of RSF1010 increase dramatically if the recipient strain, as well as the donor strain, carries a wild-type Ti plasmid and is capable of vir gene expression . Investigation of possible mechanisms that could account for this increased efficiency revealed that the VirB proteins encoded by the Ti plasmid were required . Although, with the exception of VirB1, all of the proteins that form the putative T-DNA transfer apparatus (VirB1-11, VirD4) are required for an Agrobacterium strain to serve as an RSF1010 donor, expression of only a subset of these proteins is required for the increase in conjugal transfer mediated by the recipient . Specifically, VirB5, 6, 11, and VirD4 are essential donor components but are dispensable for the increased recipient capacity . Defined point mutations in virB9 affected donor and recipient capacities to the same relative extent, suggesting that similar functions of VirB9 are important in both of these contexts.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7040 - 5
Agrobacterium VirD2 protein interacts with plant host cyclophilins; Deng W et al.; Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell . The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein . The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex . Three VirD2- and two VirE2-interacting proteins were identified . Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis . The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains . The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco . These data strongly suggest that host cyclophilins play a role in T-DNA transfer.

J Bacteriol, 1998 Jun, 180(12), 3107 - 13
An isoflavonoid-inducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots; Palumbo JD et al.; Agrobacterium tumefaciens 1D1609, which was originally isolated from alfalfa (Medicago sativa L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells . Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn5-B30 . Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame, ifeA, of a three-gene locus (ifeA, ifeB, and ifeR), which shows high homology to bacterial efflux pump operons . Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (P < or = 0.01) in competition against wild-type strain 1D1609 . Site-directed mutagenesis experiments, which produced strains I-4 (ifeA::gusA) and I-6 (ifeA::omega-Tc), confirmed the importance of ifeA for competitive root colonization . Exposure to the isoflavonoid coumestrol increased beta-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined . In the same test, coumestrol accumulation in mutant strain I-6 did not decline . Expression of the ifeA-gusA reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa . These results show that an efflux pump can confer measurable ecological benefits on A . tumefaciens in an environment where the inducing molecules are known to be present.

Genetics, 1998 Jun, 149(2), 693 - 701
Characterization of the putative transposase mRNA of Tag1, which is ubiquitously expressed in Arabidopsis and can be induced by Agrobacterium-mediated transformation with dTag1 DNA; Liu D et al.; Tag1 is an autonomous transposable element of Arabidopsis thaliana . Tag1 expression was examined in two ecotypes of Arabidopsis (Columbia and No-0) that were transformed with CaMV 35S-Tag1-GUS DNA . These ecotypes contain no endogenous Tag1 elements . A major 2.3-kb and several minor transcripts were detected in all major organs of the plants . The major transcript encoded a putative transposase of 84.2 kD with two nuclear localization signal sequences and a region conserved among transposases of the Ac or hAT family of elements . The abundance of Tag1 transcripts varied among transgenic lines and did not correlate with somatic excision frequency or germinal reversion rates, suggesting that factors other than transcript levels control Tag1 excision activity . In untransformed plants of the Landsberg ecotype, which contain two endogenous Tag1 elements, no Tag1 transcripts were detected . Agrobacterium-mediated transformation of these Landsberg plants with a defective 1.4-kb Tag1 element resulted in the appearance of full-length Tag1 transcripts from the endogenous elements . Transformation with control DNA containing no Tag1 sequences did not activate endogenous Tag1 expression . These results indicate that Agrobacterium-mediated transformation with dTag1 can activate the expression of Tag1.

Plant Mol Biol, 1998 Jun, 37(3), 581 - 5
A plant transformation vector with a minimal T-DNA II . Irregular integration patterns of the T-DNA in the plant genome; Porsch P et al.; A minimal T-DNA binary vector was used for Agrobacterium-mediated transfer of a chimeric T4 lysozyme gene located next to the left border, and transgenic potato plants which expressed T4 lysozyme protein were identified and further analysed . Frequent rearrangements of T4 lysozyme transgenes were detected . A vector derivative containing two matrix associated regions (MARs) flanking its multiple cloning site was constructed . In transgenic potato plants, reduced variability in gene expression due to position effects was detected . When either the donor vector contained MAR sequences, or when vector pPCV701 which contains a pBR322 fragment next to the left border were used, only relatively few rearrangements were observed . However, when the T4 lysozyme gene was driven by a CaMV 35S promoter modified by multiplied enhancer region carrying either 2 or 4 elements, frequent rearrangements were again obtained.

Plant Mol Biol, 1998 Jun, 37(3), 549 - 59
Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting; Maximova SN et al.; To investigate early events of Agrobacterium-mediated transformation of apple cultivars, a synthetic green fluorescent protein gene (SGFP) was used as a highly sensitive, vital reporter gene . Leaf explants from four apple cultivars ('Delicious', 'Golden Delicious', 'Royal Gala' and 'Greensleeves') were infected with Agrobacterium EHA101 harboring plasmid pDM96.0501 . Fluorescence microscopy indicated that SGFP expression was first detected 48 h after infection and quantitative analysis revealed a high T-DNA transfer rate . Plant cells with stably incorporated T-DNA exhibited cell division and developed transgenic calli, followed by formation of transgenic shoots at low frequencies . The detection of SGFP expression with an epifluorescence stereomicroscope confirmed the effectiveness of SGFP as a reporter gene for detection of very early transformation events and for screening of putative transformants . The efficiency of the transformation and regeneration process decreased ca . 10,000-fold from Agrobacterium infection to transgenic shoot regeneration, suggesting that factors other than Agrobacterium interaction and T-DNA transfer are rate-limiting steps in Agrobacterium-mediated transformation of apple.

Plant Mol Biol, 1998 May, 37(2), 287 - 96
The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent; Haldrup A et al.; The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium . The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation . The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the omega' translation enhancer sequence from tobacco mosaic virus . Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed . Efficiency may be increased by optimization . The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source . It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive . This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed . The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection.

Plant Mol Biol, 1998 May, 37(2), 275 - 85
Functional analysis of the promoter region of a maize (Zea mays L.) H3 histone gene in transgenic Arabidopsis thaliana; Atanassova R et al.; A 1023 bp fragment and truncated derivatives of the maize (Zea mays L.) histone H3C4 gene promoter were fused to the beta-glucuronidase (GUS) gene and introduced via Agrobacterium tumefaciens into the genome of Arabidopsis thaliana . GUS activity was found in various meristems of transgenic plants as for other plant histone promoters, but unexplained activity also occurred at branching points of both stems and roots . Deletion of the upstream 558 bp of the promoter reduced its activity to an almost basal expression . Internal deletion of a downstream fragment containing plant histone-specific sequence motifs reduced the promoter activity in all tissues and abolished the expression in meristems . Thus, both the proximal and distal regions of the promoter appear necessary to achieve the final expression pattern in dicotyledonous plant tissues . In mesophyll protoplasts isolated from the transformed Arabidopsis plants, the full-length promoter showed both S phase-dependent and -independent activity, like other plant histone gene promoters . Neither of the 5'-truncated nor the internal-deleted promoters were able to direct S phase-dependent activity, thus revealing necessary cooperation between the proximal and distal parts of the promoter to achieve cell cycle-regulated expression . The involvement of the different regions of the promoter in the different types of expression is discussed.

Virus Res, 1998 Jan, 53(1), 97 - 103
Nicotiana benthamiana plants transformed with the plum pox virus helicase gene are resistant to virus infection; Wittner A et al.; Nicotiana benthamiana Domin . plants were transformed with the cytoplasmic inclusion protein (CI) gene of plum pox potyvirus (PPV) to investigate, whether this non-structural protein would be able to confer resistance . The CI protein is an RNA helicase, which contains a conserved nucleotide binding motif (NTBM) and plays an important role in viral replication . Two gene constructions were developed for plant transformation . The first contains the original coding sequence of the CI gene under the control of 35S-promoter and nos terminator signal, the second is mutated in the NTBM region . Several transgenic plant lines were obtained following Agrobacterium tumefaciens-mediated transformation . The integration of the viral genes into the plant genome was confirmed using the polymerase chain reaction and the transgene derived mRNAs were detected by Northern blot hybridization . The CI protein in the transgenic plants could not be detected by Western blot analyses . One transgenic line containing the mutated CI gene remained completely symptomless after PPV infection, indicating that the putative defective helicase gene was capable of eliciting virus resistance.

J Bacteriol, 1998 Jun, 180(11), 2866 - 74
Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici; Mavingui P et al.; Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli . Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici . To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R . tropici CFN299 . Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture . The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens . The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1 . Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors . Strikingly, the conjugal transfer of the amplicon into a plasmidless A . tumefaciens strain confers on the transconjugant the ability to produce R . tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R . tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.

J Bacteriol, 1998 May, 180(10), 2749 - 55
Unconventional genomic organization in the alpha subgroup of the Proteobacteria; Jumas-Bilak E et al.; Pulsed-field gel electrophoresis was used to analyze the genomic organization of 16 bacteria belonging or related to the family Rhizobiaceae of the alpha subgroup of the class Proteobacteria . The number and sizes of replicons were determined by separating nondigested DNA . Hybridization of an rrn gene probe was used to distinguish between chromosomes and plasmids . Members of the genus Agrobacterium all possess two chromosomes, and each biovar has a specific genome size . As previously demonstrated for Agrobacterium tumefaciens C58, the smaller chromosomes of Agrobacterium biovar 1 and Agrobacterium rubi strains appear to be linear . The genomes of Rhizobium strains were all of similar sizes but were seen to contain either one, two, or three megareplicons . Only one chromosome was present in the member of the related genus Phyllobacterium . We found one or two chromosomes in Rhodobacter and Brucella species, two chromosomes in Ochrobactrum anthropi, and one chromosome in Mycoplana dimorpha and Bartonella quintana; all of these genera are related to the Rhizobiaceae . The presence of multiple chromosomes is discussed from a phylogenetic and taxonomic point of view.

J Bacteriol, 1998 May, 180(10), 2711 - 7
Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens; Lai EM et al.; Previous studies have implicated the obligatory requirement for the vir regulon (or "virulon") of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells . The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system . Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli . The F pilus is composed of TraA pilin subunits derived from TraA propilin . In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses . The processed VirB2 protein is present exocellularly on medium on which induced A . tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody . Exocellular VirB2 is produced abundantly at 19 degreesC as compared with 28 degreesC, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K . J . Fullner, L . C . Lara, and E . W . Nester, Science 273:1107-1109, 1996) . Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell . The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation . The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the "T pilus" of A . tumefaciens.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5293 - 8
Agrobacterium transcriptional regulator Ros is a prokaryotic zinc finger protein that regulates the plant oncogene ipt; Chou AY et al.; Virulence genes of Agrobacterium tumefaciens are under the control of positive and negative transcriptional regulators . We found that the transcriptional regulator Ros controls expression of the plant oncogene ipt, which encodes isopentenyl transferase, in A . tumefaciens . This enzyme is involved in biosynthesis of the plant growth hormone cytokinin in the host plant . An ipt promoter::cat reporter gene fusion showed a 10-fold increase in ipt promoter activity in A . tumefaciens ros mutant strains when compared with wild type . Also, increased levels (10- to 20-fold) of isopentenyl adenosine, the product of the reaction catalyzed by isopentenyl transferase, were detected in ros mutant strains . In vitro studies using purified Ros showed it binds directly to the ipt promoter . Analysis of the deduced Ros amino acid sequence identified a novel type of C2H2 zinc finger . In Ros the peptide loop spacing of the zinc finger is 9 amino acids as opposed to the invariant 12 amino acids in the classical C2H2 motif . Site-directed mutagenesis of Cys-82 and His-92 in this motif showed that these residues are essential for Zn2+ and DNA binding activities of Ros . The existence of such a regulator in Agrobacterium may be due to horizontal interkingdom retrotransfer of the ros gene from plant to bacteria.

Mol Plant Microbe Interact, 1998 Jun, 11(6), 449 - 57
Agrobacterium tumefaciens transformation and cotransformation frequencies of Arabidopsis thaliana root explants and tobacco protoplasts; De Buck S et al.; In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains . The transformation frequency of unselected A . thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18% . The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used . Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells . Molecular analysis of cocultivated N . tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA . Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants . Remarkably, 10 to 30% of the selected A . thaliana shoots or progenies lost the T-DNA marker they were selected on . As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs . In conclusion, a significant part of the T-DNAs is lost from the transformed cells.

Transgenic Res, 1998 Mar, 7(2), 77 - 84
Insect resistance of transgenic tobacco expressing an insect chitinase gene; Ding X et al.; Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded . Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase . This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation . A truncated but enzymatically active chitinase was present in plants expressing the gene . Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage . Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene . In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics . However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls . Foliar damage was also reduced . Plants expressing an insect chitinase gene may have agronomic potential for insect control.

Arch Microbiol, 1998 May, 169(5), 381 - 6
Characterization of a new Agrobacterium tumefaciens strain from alfalfa; Palumbo JD et al.; Agrobacterium tumefaciens strain 1D1609 is reported here as the first field isolate from alfalfa (Medicago sativa L.) . Unlike well-characterized A . tumefaciens strains such as C58 and Ach5, strain 1D1609 is highly virulent on alfalfa and has a distinctive host range . Interestingly, strain 1D1609 is naturally resistant to kanamycin and spectinomycin . The Ti plasmid in strain 1D1609 is an octopine-type; thus, tumors formed by strain 1D1609 synthesize octopine, which is utilized by the bacterium as a sole carbon source . Reciprocal exchange of Ti plasmids between strains 1D1609 and C58 showed that both chromosomal and Ti plasmid genes in strain 1D1609 contribute specifically to tumor formation on alfalfa . In addition, the nondormant CUF101 alfalfa cultivar from which strain 1D1609 was isolated was significantly more susceptible to all Agrobacterium strains tested than was the dormant Agate cultivar.

Antonie Van Leeuwenhoek, 1998 Jan, 73(1), 117 - 26
Origin and evolution of plasmids; Kado CI; Studies on the origin and evolution of plasmids may provide valuable insights on the promiscuous nature of DNA . The first examples of the selfish nature of nucleic acids are exemplified by primordial oligoribonucleotides which evolved into primitive replicons . The propagation of these molecules were likely patterned after the current viral RNA ribozymes, which have been recently shown to possess RNA synthesizing and template mediated polymerizing capabilities in the absence of proteins . The parasitic nature of nucleic acids is depicted by satellite nucleic acid molecules associated with viruses . The satellite of adenovirus and tobacco ringspot virus serve as established examples: they contain no open reading frames . Comparative analysis of the replication origins of virions and plasmids show them to be conserved, originating from the simplest autocatalytic replicon to highly complex and evolved plasmids, replicating by a rolling circle mechanism . The eventual association of proteins with nucleic acids provided added efficiency and protective advantages for molecular perpetuation . The promiscuous and selfish nature of plasmids is demonstrated by their ability to genetically engineer their host so that the host cell is best able to cope and survive in hostile environments . Survival of the host ensures survival of the plasmid . Sequestering of genes by plasmids occurs when the environmental conditions negatively affect the host . The sequestering mechanism is fundamental and forms the outreach mechanisms to generate and propagate macromolecules of increasing size when necessary for survival . The level of sophistication of plasmids increases with the addition of new genes such as those that allow the host to occupy a specific environment normally inhospitable to the host cell . The vast range of plasmid types which have obtained genes interchangeably reflect the levels of sophistication achieved by these macromolecules . The Ti plasmid in Agrobacterium tumefaciens and the pSym and accessory plasmids in Rhizobium illustrate the level of complexity attained by replicons.

Plant Physiol, 1998 Apr, 116(4), 1367 - 77
Characterization of a gene for spinach CAP160 and expression of two spinach cold-acclimation proteins in tobacco; Kaye C et al.; The cDNA sequence for CAP160, an acidic protein previously linked with cold acclimation in spinach (Spinacia oleracea L.), was characterized and found to encode a novel acidic protein of 780 amino acids having very limited homology to a pair of Arabidopsis thaliana stress-regulated proteins, rd29A and rd29B . The lack of similarity in the structural organization of the spinach and Arabidopsis genes highlights the absence of a high degree of conservation of this cold-stress gene across taxonomic boundaries . The protein has several unique motifs that may relate to its function during cold stress . Expression of the CAP160 mRNA was increased by low-temperature exposure and water stress in a manner consistent with a probable function during stresses that involve dehydration . The coding sequences for CAP160 and CAP85, another spinach cold-stress protein, were introduced into tobacco (Nicotiana tabacum) under the control of the 35S promoter using Agrobacterium tumefaciens-based transformation . Tobacco plants expressing the proteins individually or coexpressing both proteins were evaluated for relative freezing-stress tolerance . The killing temperature for 50% of the cells of the transgenic plants was not different from that of the wild-type plants . As determined by a more sensitive time/temperature kinetic study, plants expressing the spinach proteins had slightly lower levels of electrolyte leakage than wild-type plants, indicative of a small reduction of freezing-stress injury . Clearly, the heterologous expression of two cold-stress proteins had no profound influence on stress tolerance, a result that is consistent with the quantitative nature of cold-stress-tolerance traits.

Plant Physiol, 1998 Apr, 116(4), 1307 - 13
Effect of ATP sulfurylase overexpression in bright yellow 2 tobacco cells . Regulation Of atp sulfurylase and SO4(2-) transport activities; Hatzfeld Y et al.; To determine if the ATP sulfurylase reaction is a regulatory step for the SO4(2-)-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 35S promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells . The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody . The molecular mass of this polypeptide agreed with that for the overexpressed mature protein . ATP sulfurylase overexpression had no effect on {35S}SO4(2-) influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type . There were also no differences in cell growth or sensitivity to SeO4(2-) (a toxic SO4(2-) analog) between transgenic and wild-type cells . We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism.

Vox Sang, 1998, 74(3), 148 - 55
Application of the human hepatitis B virus core antigen from transgenic tobacco plants for serological diagnosis; Tsuda S et al.; BACKGROUND AND OBJECTIVES: The aim was to produce HBcAg from plants more cheaply than can be done by other currently available means, and to apply such antigen to immunoassay procedures for pretransfusion testing of donor blood . MATERIALS AND METHODS: Transgenic Nicotiana tabacum cv . SR-1 plants expressing the human hepatitis B virus (HBV) core antigen (HBcAg) gene were generated by Agrobacterium-mediated transformation . The recombinant product, called tHBcAg, can assemble itself into a spherical particle with a diameter of 25 to 30 nm, and can maintain two antigenic determinants of HBcAg, namely HBc/alpha and HBc/beta . Partly purified tHBcAg was used in the hemagglutination-inhibition (HI) test, as routinely used by the Japanese Blood Center, to test a panel of 524 blood units taken from HBV-positive donors . RESULTS: In the HI test, tHBcAg showed serologic properties comparable to that from Escherichia coli, the standard antigen used in the Japanese Blood Center . CONCLUSIONS: Transgenic plants can produce reagents for serologic testing and perhaps even such medical materials as oral vaccines.

Mol Microbiol, 1998 Apr, 28(1), 37 - 53
Analyses of the cag pathogenicity island of Helicobacter pylori; Akopyants NS et al.; Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene . Recent studies showed that the cagA gene lies near the right end of a approximately 37kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA+ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638 . In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left 'cagII' half of this PAI . Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis ('Ptl'), Agrobacterium tumefaciens ('Vir'), and conjugative plasmids ('Tra') that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure . To the left of cagII in this cosmid were genes for homologues of HsIU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions . Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells . Comparisons among H . pylori strains indicated that cag PAI gene content and arrangement are rather well conserved . We also identified two genome rearrangements with end-points in the cag PAI . One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others) . Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605 . Our results are discussed in terms of how cag-encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.

J Antibiot (Tokyo), 1997 Nov, 50(11), 916 - 8
Hydroxyakalone, a novel xanthine oxidase inhibitor produced by a marine bacterium, Agrobacterium aurantiacum; Izumida H et al.; A new xanthine oxidase inhibitor named hydroxyakalone was isolated from the culture broth of a marine bacterium Agrobacterium aurantiacum N-81106 . Structure of hydroxyakalone was determined to be 4-amino-1H-pyrazolo{3,4-d}pyrimidine-3-one-6-ol by the spectral studies of hydroxyakalone and its permethyl derivative . The concentration to induce 50% inhibition (IC50) was 4.6 microM against xanthine oxidase.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2729 - 34
Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase; Vergunst AC et al.; In this study Agrobacterium tumefaciens transferred DNA (T-DNA) was targeted to a chromosomally introduced lox site in Arabidopsis thaliana by employing the Cre recombinase system . To this end, Arabidopsis target lines were constructed which harboured an active chimeric promoter-lox-cre gene stably integrated in the plant genome . A T-DNA vector with a promoterless lox -neomycin phosphotransferase (nptII) fusion was targeted to this genomic lox site with an efficiency of 1.2-2.3% of the number of random events . Cre-catalyzed site-specific recombination resulted in restoration of nptII expression by translational fusion of the lox-nptII sequence in the integration vector with the transcription and translation initiation sequences present at the target site, allowing selective enrichment on medium containing kanamycin . Simultaneously, the coding sequence of the Cre recombinase was disconnected from these same transcription and translation initiation signals by displacement, aimed at preventing the efficient reversible excision reaction . Of the site-specific recombinants, 89% were the result of precise integration . Furthermore, approximately 50% of these integrants were single copy transformants, based on PCR analysis . Agrobacterium T-DNA, which is transferred to plant cells as a single-stranded linear DNA structure, is in principle incompatible with Cre-mediated integration . Nevertheless, the results presented here clearly demonstrate the feasibility of the Agrobacterium -mediated transformation system, which is generally used for transformation of plants, to obtain site-specific integration.

Biochimie, 1998 Jan, 80(1), 87 - 94
Purification of two lectins from a nopalin Agrobacterium tumefaciens strain; Kang HC et al.; Lectins were evidenced on the surface of one Agrobacterium tumefaciens wild strain (82,139) by agglutination test and neoglycoprotein labelling . Bacteria were incubated in the presence of various fluorescein-labelled neoglycoproteins and the binding was assessed by a fluorimetric method . Among the fluorescein-labelled neoglycoproteins tested, the one bearing alpha-D-galactosyl residues was the most efficient . The labelling was optimal at pH 5.0 and naught at pH above 7 . The binding was specifically inhibited by homologous fluorescein-free neoglycoproteins . A galactoside-specific lectin was purified to homogeneity by affinity chromatography on agarose-A4 substituted with alpha-D-galactopyranosyl residues . Upon polyacrylamide gel electrophoresis, a single band (M(r) 58,000) was detected . This alpha-D-galactoside-specific lectin agglutinated preferentially human B red blood cells at pH 5.0 . Another lectin specific for alpha-L-rhamnoside (M(r) 40,000) not retained on the immobilised galactose was purified by affinity chromatography on alpha-L-rhamnosyl substituted agarose-A4 . This L-rhamnoside-specific lectin preferentially agglutinated horse erythrocytes . On the basis of their M(r) and on their sugar specificity, these two lectins are novel lectins with regard to the known sugar-binding proteins present in the Rhizobiaceae family: Agrobacterium, Rhizobium or Bradyrhizobium strains.

Nucleic Acids Symp Ser, 1997, (37), 161 - 2
Genome structure of pTi-SAKURA (II): genetic map constructed by complete DNA sequencing; Suzuki K et al.; Ti plasmid (pTi-SAKURA) DNA isolated from an agrobacterium pathogenic against Japanese cherry trees were completely sequenced by primer walking with PCR subcloning . Typical genes including transfer DNA (T-DNA), nopaline utilizing genes, trb genes, traI, rep genes, tra genes, acc and vir genes were assigned in this order to pTi-SAKURA . Between the rep genes and tra genes, we found a large region which essentially lacks homology to any sequences in DNA databases . By amino acid sequence search, we could pick up several ORFs which are homologous with genes putatively capable to enhance interaction between agrobacteria and plants.

Nucleic Acids Symp Ser, 1997, (37), 159 - 60
Genome structure of pTi-SAKURA (I): strategy for DNA sequencing of a Japanese cherry-Ti plasmid; Hattori Y et al.; We isolated a plasmid from a bacterium Agrobacterium tumefaciens, which had been found in a crown gall tumor on a Japanese cherry tree SAKURA and designated it pTi-SAKURA . For complete DNA sequencing, we constructed a DNA library in lambda phage vector and developed a sequencing method by primer walking with long PCR and a PCR subcloning technique for long insert DNA.

Nucleic Acids Symp Ser, 1997, (37), 157 - 8
Construction of a novel conjugative plasmid harboring a GFP reporter gene and its introduction into animal cells by transfection and trans-kingdom conjugation; Yoshida K et al.; We have established a novel method of gene introduction into eukaryotic cells by trans-kingdom conjugation between Escherichia coli/Agrobacterium tumefaciens bacteria and yeasts . To expand this to animal cells, we have constructed a novel conjugative plasmid, pBASGreen which contains SV40-ori/promoter, pUC-ori, IncQ type oriT/mob, Apr and Neor genes with GFP (green fluorescent protein) gene as a reporter . The introduction into COS1 and NIH3T3 animal cultured cells by conventional transfection was easily detected by fluorescence of the GFP gene product under a fluorescent microscope and the transfectants were effectively selected by the Neor marker . By the action of oriT/mob in the presence of tra genes on a helper plasmid, pBASGreen was directly mobilizable from E . coli into animal cells by trans-kingdom conjugation.

Acta Biochim Pol, 1997, 44(4), 819 - 25
Characterization of two lipopolysaccharide types isolated from Rhizobium galegae; Rasanen LA et al.; Lipopolysaccharides (LPS) of Rhizobium galegae, a symbiotically nitrogen-fixing species of root-nodule bacteria, were isolated by the phenol-water method from strain HAMBI 1461, the LPS of which resembled enterobacterial smooth type LPS, and from strains HAMBI 1174 and HAMBI 1208, the LPSs of which resembled rough type LPS . The results of PAGE analysis of LPSs, Bio-Gel P2 gel filtration of polysaccharide fractions and the presence of deoxysugars and 4-O-methyl-deoxysugar both in the rough and smooth LPSs suggested that rough LPS contained a short O-antigenic polysaccharide for which we propose the name short O-chain LPS . Accordingly, the smooth LPS is called long O-chain LPS . Despite of the differences in the structure of LPS of R . galegae, all strains were equally effective in nodulating their hosts . The short O-chain LPS of R . galegae showed many features similar to those of phylogenetically related agrobacteria.

J Antibiot (Tokyo), 1998 Jan, 51(1), 64 - 7
Two marine Agrobacterium producers of sesbanimide antibiotics; Acebal C et al.; Sesbanimides are cytotoxic compounds, originally isolated in 1983 from seeds of the leguminous plants Sesbania drummondii and Sesbania punicea . In this paper we describe the bacterial production of sesbanimides by two "marine Agrobacterium"; strain PH-103 which produces Sesbanimide-A and strain PH-A034C which produces Sesbanimide-C . The isolation and taxonomy of the producing microorganisms, fermentation and isolation of sesbanimides are reported.

Microbiology, 1998 Apr, 144 ( Pt 4), 947 - 54
Efficient conversion of 5-substituted hydantoins to D-alpha-amino acids using recombinant Escherichia coli strains; Grifantini R et al.; D-Amino acids, important intermediates in the production of semisynthetic penicillins and cephalosporins, are currently prepared from the corresponding hydantoins using bacterial biomass containing two enzymes, hydantoinase and carbamylase . These enzymes convert the hydantoins first into carbamyl derivatives and then into the corresponding D-amino acids . In an attempt to select more efficient biocatalysts, the hydantoinase and carbamylase genes from Agrobacterium tumefaciens (formerly A . radiobacter) were cloned in Escherichia coli . The genes were assembled to give two operon-type structures, one having the carbamylase gene preceding the hydantoinase gene and the other with the carbamylase gene following the hydantoinase gene . The recombinant strains stably and constitutively produced the two enzymes and efficiently converted the corresponding hydantoins into p-hydroxyphenylglycine and phenylglycine . The order of the genes within the operon and the growth temperature of the strains turned out to be important for both enzyme and D-amino acid production . The configuration with the carbamylase gene preceding the hydantoinase gene was the most efficient one when the biomass was grown at 25 degrees C rather than 37 degrees C . This biomass produced D-amino acid twice as efficiently as the industrial strain of A . tumefaciens . The efficiency was found to be correlated with the level of carbamylase produced, indicating that the concentration of this enzyme is the rate-limiting factor in D-amino acid production under the conditions used on an industrial scale.

Eur J Biochem, 1998 Apr 1, 253(1), 173 - 83
Characterisation of a catabolic epoxide hydrolase from a Corynebacterium sp; Misawa E et al.; The epoxide hydrolase (EH) from Corynebacterium sp . C12, which grows on cyclohexene oxide as sole carbon source, has been purified to homogeneity in two steps, involving anion exchange followed by hydrophobic-interaction chromatography . The purified enzyme is multimeric (probably tetrameric) with a subunit size of 32,140 Da . The gene encoding Corynebacterium EH was located on a 3.5-kb BamHI fragment of C12 chromosomal DNA using a DNA probe generated by PCR using degenerate primers based on the N-terminal and an internal amino acid sequence . Sequencing and database comparison of the predicted amino acid sequence of Corynebacterium EH shows that it is similar to mammalian and plant soluble EH, and the recently published sequence of epichlorohydrin EH from Agrobacterium radiobacter AD1 {Rink, R., Fennema, M., Smids, M., Dehmel, U . & Janssen, D . B . (1997) J . Biol . Chem . 272, 14650- 14657), particularly around the catalytic site . All of these proteins belong to the alpha/beta-hydrolase-fold family of enzymes . Similarity to the mammalian microsomal EH is weaker.

Mol Plant Microbe Interact, 1998 May, 11(5), 429 - 33
The presence and characterization of a virF gene on Agrobacterium vitis Ti plasmids; Schrammeijer B et al.; Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca . The presence of a virF locus on the octopine Ti plasmid makes N . glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant . Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plasmids pTiAT1, pTiAT66a, and pTiAT66b . On the octopine Ti plasmids from A . tumefaciens the virF gene is located between the virE locus and the left border of the T-region . In contrast, the virF gene on Ti plasmids of A . vitis is located at the very left end of the vir-region near the virA locus . The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da . Comparison showed that the virF gene from A . vitis strain Ag57 is almost identical to that from A . tumefaciens octopine strains . The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region . The VirF protein from pTiAg57 can complement octopine A . tumefaciens strains deleted for virF as shown by tumor formation on N . glauca.

Mol Plant Microbe Interact, 1998 May, 11(5), 335 - 42
A T-DNA from the Agrobacterium tumefaciens limited-host-range strain AB2/73 contains a single oncogene; Otten L et al.; Agrobacterium tumefaciens strain AB2/73 isolated from Lippia canescens has been described as a limited-host-range strain . Its tumor-inducing (Ti) plasmid has been found to lack DNA homology to known T-DNAs (L . Unger, S . F . Ziegler, G . A . Huffman, V . C . Knauf, R . Peet, L . W . Moore, M . P . Gordon, and E . W . Nester . J . Bacteriol . 164:723-730, 1985) . We have isolated a T-DNA from AB2/73 by using a heterologous border sequence as a probe . The AB2/73 T-DNA sequence (3,504 bp) is flanked by canonical border sequences, has no detectable DNA homology with other T-DNAs, and contains only two genes: lsn (Lippia strain nopaline synthaselike gene) and lso (Lippia strain oncogene) . The lso gene induces nondifferentiating tumors on a limited number of hosts when transferred by a Ti plasmid from a wide-host-range strain . Part of the predicted Lso protein is weakly homologous to other Agrobacterium oncoproteins encoded by rolB, rolB, orf13, gene e, gene 5, and gene 3' . A 28-kb fragment corresponding to the virA to virE region was cloned by using a heterologous vir fragment as probe . The AB2/73 vir region is homologous to most of the C58 virulence region; however, the virA gene is most related to the virA gene of the Agrobacterium vitis limited-host-range strain Ag162.

Gene, 1998 Apr 14, 210(2), 307 - 14
Characterization of an unusual sensor gene (virA) of Agrobacterium; Lee YW et al.; Previous studies have shown that the virulence(vir) genes of Agrobacterium tumefaciens strain KU12 are induced by a unique set of phenolic compounds that are non-functional in most strains of Agrobacterium . Further, strain KU12 is not induced by phenolic compounds that induce the vir genes in other strains . Previous studies have shown that these differences in inducing activity result from differences in the sensor protein for these signal molecules, the VirA protein . To gain some understanding of the basis for these differences in sensing ability, we sequenced the entire virA locus of pTiKU12, including its promoter region and compared this sequence with five different published virA sequences that respond in different ways to inducing compounds . The virA gene of KU12 is composed of an open single reading frame coding for 851 aa . At the aa level, the VirA protein of pTiKU12 is 45, 45, 49, 49 and 64% identical to the VirA proteins from pTiA6, pTi15955, pRiA4, pTiC58 and pTiAg162, respectively . The transcription start sites of pTiKU12 and pTiA6 virA genes differ significantly when mapped by primer extension . Unlike all other vir genes, except the virA gene of pTiAg162, pTiKU12 virA is constitutively expressed, and its synthesis is not induced by phenolic compounds . The lack of induction is accounted for by the fact that the promoter region does not have the conserved VirG-binding dodecadeoxynucleotide sequence (vir-box) that was previously identified in all promoter regions of inducible vir genes.

J Bacteriol, 1998 May, 180(9), 2402 - 8
Regulatory conservation and divergence of sigma32 homologs from gram-negative bacteria: Serratia marcescens, Proteus mirabilis, Pseudomonas aeruginosa, and Agrobacterium tumefaciens; Nakahigashi K et al.; The heat shock response in Escherichia coli is mediated primarily by the rpoH gene, encoding sigma32, which is specifically required for transcription of heat shock genes . A number of sigma32 homologs have recently been cloned from gram-negative bacteria that belong to the gamma or alpha subdivisions of the proteobacteria . We report here some of the regulatory features of several such homologs (RpoH) expressed in E . coli as well as in respective cognate bacteria . When expressed in an E . coli delta rpoH strain lacking its own sigma32, these homologs activated the transcription of heat shock genes (groE and dnaK) from the start sites normally used in E . coli . The level of RpoH in Serratia marcescens and Pseudomonas aeruginosa cells was very low at 30 degrees C but was elevated markedly upon a shift to 42 degrees C, as found previously with E . coli . The increased RpoH levels upon heat shock resulted from both increased synthesis and stabilization of the normally unstable RpoH protein . In contrast, the RpoH level in Proteus mirabilis was relatively high at 30 degrees C and increased less markedly upon heat shock, mostly by increased synthesis; this sigma32 homolog was already stable at 30 degrees C, and little further stabilization occurred upon the shift to 42 degrees C . The increased synthesis of RpoH homologs in all these gamma proteobacteria was observed even in the presence of rifampin, suggesting that the induction occurred at the level of translation . Thus, the basic regulatory strategy of the heat shock response by enhancing the RpoH level is well conserved in the gamma proteobacteria, but some divergence in the actual mechanisms used occurred during evolution.

Proc Soc Exp Biol Med, 1998 May, 218(1), 83 - 9
Absence of mycoplasmal gene in malignant mammalian cells transformed by chronic persistent infection of mycoplasmas; Zhang B et al.; Chronic persistent infections by mycoplasmas induced malignant transformation of C3H mouse embryo cells that normally had never been reported to undergo spontaneous transformation . This mycoplasma-mediated oncogenic process had a long latency (more than 7 weeks of continuous mycoplasmal infection) and showed a multistage progression characterized by reversibility (at least up to 11 weeks of mycoplasmal infection) and irreversibility of malignant properties upon removal of the mycoplasma from culture . Further prolonged infections (18 weeks) by Mycoplasma fermentans or M . penetrans resulted in permanent transformation of these C3H cells that no longer required the continued presence of the transformation-inducing mycoplasmas in cultures to retain their malignant properties . Previous studies of viral oncogenesis revealed that virus-transformed cells always had viral gene(s) present . Integration of viral gene(s) apparently played an important role in the process of oncogenesis . In this study, we examined if the continued presence of any mycoplasmal gene(s) in mammalian cells, in whatever form, was also crucial in causing malignant cell transformation . Representational difference analysis (RDA) was a recently developed powerful technique to compare differences between two complex genomes . In the RDA system, subtractive and kinetic enrichment was used to purify and isolate restriction endonuclease gene fragment(s) of mycoplasmal origin, presumably present only in mycoplasma-transformed C3H cells, but not in nonmycoplasma-exposed control C3H cells . After three rounds of subtractive hybridization following PCR enrichment for each of three different restriction enzymes DNA digests, no gene fragment of mycoplasmal origin was amplified or identified in the permanently transformed C3H cells . Differing from tumorigenesis in animal cells induced by most oncogenic viruses or in plant cells induced by Agrobacteria, mycoplasmas evidently did not cause malignant transformation by integrating their gene(s) into the mammalian cell genome.

Biosci Biotechnol Biochem, 1998 Mar, 62(3), 438 - 42
Novel bioactive oxazolomycin isomers produced by Streptomyces albus JA3453; Kanzaki H et al.; Two novel oxazolomycin isomers, oxazolomycins B (2) and C (3), were isolated from the fermentation broth of an oxazolomycin-producing strain, Streptomyces albus JA3453 . Both compounds are geometrical isomers of oxazolomycin (1), the configurations of their triene moieties being (4'E, 6'E, 8'E) (2) and (4'Z, 6'E, 8'E) (3) while that of oxazolomycin (1) is (4'Z, 6'Z, 8'E) . Compounds 2 and 3 exhibited potent inhibitory activity against crown gall formation with the same MIC (0.8 microgram/disk) as oxazolomycin . Compounds 2 and 3 showed no antibacterial activity against Agrobacterium tumefaciens, in contrast to oxazolomycin which has specific anti-A . tumefaciens activity.

Mol Cells, 1998 Feb 28, 8(1), 49 - 53
The role of inverted repeat (IR) sequence of the virE gene expression in Agrobacterium tumefaciens pTiA6; Jeon GA et al.; Agrobacterium tumefaciens pTiA6 virE promoter has a vir box, an inverted repeat (IR) sequence, a putative -35 region and a consensus -10 region . To study how the IR sequence of the virE promoter plays a role in virE gene expression, various mutants were constructed by base substitution and deletion in the virE promoter region . Substitution of the 3'-end region of the IR sequence, 5'-TCCGTTTCAA-3' to 5'-GCGGCCGCTC-3' displayed 2.6% of the native virE promoter activity . A deletion mutant of 5'-CGTTTCAA-3' on the 3'-end region of the IR sequence expressed 6% of the native virE promoter activity . These mutational analyses demonstrated that the IR sequence of the virE promoter plays a role as a cis-acting element in virE expression.

Cell Biol Int, 1997 Sep, 21(9), 595 - 600
RolB expression pattern in the early stages of carrot somatic embryogenesis; Di Cola A et al.; The pattern of expression of the rolB gene, derived from the T-DNA of the plant pathogen Agrobacterium rhizogenes, has been investigated during the early stages of somatic embryo formation in suspension cultures of carrot (Daucus carota L.) . The reporter gene GUS (Escherichia coli beta-glucuronidase), under transcriptional control of full-length rolB promoter region, has been utilized in order to evaluate both qualitative and quantitative variations in the expression pattern . Fluorimetric measurements point to the developmental regulation of the gene, while results from histochemical analysis indicate that the promoter of rolB is firstly activated in the central (core) region of the globular embryos.

Sci Prog, 1998, 81 ( Pt 1), 69 - 80
Recent advances in biology: intercellular communication and quorum sensing in micro-organisms; Hussain NH et al.; N-acyl-L-homoserine lactones are involved as intercellular signalling agents controlling a wide range of physiological responses in Gram-negative bacteria . They function especially in vibrios, pseudomonads and erwinias as well as in Rhizobium and Agrobacterium spp, particularly where the bacteria are in symbiotic or parasitic relationships with higher organisms . Several Gram-negatives, such as Escherichia coli, do not, however, appear to produce or respond to AHLs and they may have other intercellular signalling molecules . The present review reports that several stress related responses in E . coli can be induced by supernatant fluids from cultures which have already induced the response . In some cases at least, the active agents in the supernatant fluids are proteins rather than AHLs.

Mol Gen Genet, 1998 Mar, 257(5), 561 - 7
The use of a thermostable beta-glucanase gene from Clostridium thermocellum as a reporter gene in plants; Piruzian ES et al.; In order to take advantage of the high thermostability of its product, beta-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants . The coding region of the licB gene was truncated at both ends . The truncated enzyme retained its activity and thermostability . The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2' gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase . Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants . The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles . The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.

Appl Environ Microbiol, 1998 Apr, 64(4), 1283 - 9
Characterization of the dominant and rare members of a young Hawaiian soil bacterial community with small-subunit ribosomal DNA amplified from DNA fractionated on the basis of its guanine and cytosine composition; Nusslein K et al.; The small-subunit ribosomal DNA (rDNA) diversity was found to be very high in a Hawaiian soil community that might be expected to have lower diversity than the communities in continental soils because the Hawaiian soil is geographically isolated and only 200 years old, is subjected to a constant climate, and harbors low plant diversity . Since an underlying community structure could not be revealed by analyzing the total eubacterial rDNA, we first fractionated the DNA on the basis of guanine-plus-cytosine (G + C) content by using bisbenzimidazole and equilibrium centrifugation and then analyzed the bacterial rDNA amplified from a fraction with a high biomass (63% G + C fraction) and a fraction with a low biomass (35% G + C fraction) . The rDNA clone libraries were screened by amplified rDNA restriction analysis to determine phylotype distribution . The dominant biomass reflected by the 63% G + C fraction contained several dominant phylotypes, while the community members that were less successful (35% G + C fraction) did not show dominance but there was a very high diversity of phylotypes . Nucleotide sequence analysis revealed taxa belonging to the groups expected for the G + C contents used . The dominant phylotypes in the 63% G + C fraction were members of the Pseudomonas, Rhizobium-Agrobacterium, and Rhodospirillum assemblages, while all of the clones sequenced from the 35% G + C fraction were affiliated with several Clostridium assemblages . The two-step rDNA analysis used here uncovered more diversity than can be detected by direct rDNA analysis of total community DNA . The G + C separation step is also a way to detect some of the less dominant organisms in a community.

Appl Environ Microbiol, 1998 Apr, 64(4), 1226 - 9
Production of the carotenoids lycopene, beta-carotene, and astaxanthin in the food yeast Candida utilis; Miura Y et al.; The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, beta-carotene, and astaxanthin . The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum . The carotenoid biosynthesis genes were individually modified based on the codon usage of the C . utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C . utilis under the control of the constitutive promotes and terminators derived from C . utilis . The resultant yeast strains accumulated lycopene, beta-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively . This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2767 - 72
Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer; Cheng X et al.; Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation . The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and beta-glucuronidase, respectively . These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific delta-endotoxins . The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques . Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants . Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.

Plant Cell, 1998 Mar, 10(3), 427 - 34
The transposition frequency of Tag1 elements is increased in transgenic Arabidopsis lines; Bhatt AM et al.; Tag1 was identified as a highly active endogenous transposable element in transgenic Arabidopsis thaliana Landsberg erecta plants carrying the maize transposable element Activator (Ac) . Here, we describe experiments designed to determine the basis for the high activity of Tag1 . The frequency of transposition of Tag1 elements was compared in lines containing or lacking Ac transposase to assess the effect of Ac transposase on Tag1 activity . Three populations of nontransgenic plants, including nontransformed regenerants, were also analyzed . The high level of activity of Tag1 did not correlate with the presence or absence of Ac transposase but was significantly higher in transgenic lines . This result was maintained through at least six generations after transformation . These data suggest that Tag1 transposition is stimulated by processes that occur during the Agrobacterium transformation and that thereafter remain active . Two Tag1 elements are tightly linked in the Landsberg erecta genome and map to the lower arm of chromosome 1 . Tag1 elements were found in only a few A . thaliana ecotypes but were present in four other Arabidopsis species.

Biochem J, 1998 Feb 15, 330 ( Pt 1), 203 - 9
Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp . beta-glucosidase with a cysteine residue; Lawson SL et al.; Retaining glycosidases achieve the hydrolysis of glycosidic bonds through the assistance of two key active-site carboxyls . One carboxyl functions as a nucleophile/leaving group, and the other acts as the acid-base catalyst . It has been suggested that a cysteine residue could fulfil the role of the active site nucleophile {Hardy and Poteete (1991) Biochemistry 30, 9457-9463} . To test the validity of this proposal, a kinetic evaluation was conducted on the active-site nucleophile cysteine mutant (Glu-358-->Cys) of the retaining beta-glucosidase from Agrobacterium sp . The Glu-358-->Cys mutant was able to complete the first step (glycosylation) of the enzymic mechanism, forming a covalent glycosyl-enzyme intermediate, but the rate constant for this step was decreased to 1/10(6) of that of the native enzyme . The subsequent hydrolysis (deglycosylation) step was also severely affected by the replacement of Glu-358 with a cysteine residue, with the rate constant being depressed to 1/10(7) or less . Thus Cys-358 functions inefficiently in both the capacity of catalytic nucleophile and leaving group . On the basis of these results it seems unlikely that the role of the active-site nucleophile in retaining glycosidases could successfully be filled by a cysteine residue.

Plant Mol Biol, 1998 Mar, 36(5), 803 - 8
A putative rolB gene homologue of the Agrobacterium rhizogenes TR-DNA has different morphogenetic activity in tobacco than rolB; Lemcke K et al.; Agrobacterium rhizogenes strains of the agropine type harbor on their Ri-plasmid two T-DNAs, a left TL-DNA and a right TR-DNA . The rolB gene of the TL-DNA is the major factor in the pathogenesis of the hairy-root disease and its constitutive expression interfere profoundly with plant morphogenesis . We have tested whether the expression of its sequence related putative homologue from the TR-DNA (rolBTR) may cause also bacterial virulence or affect plant development . Unlike rolB, rolBTR is unable to induce root formation on tobacco leaf discs . Tobacco plants expressing a chimeric 35S::rolBTR gene have reduced stature, off-shoots at the stem base and bent and wrinkled leaves with epinastic growth . 14 N-terminal amino acids which are absent in the rolB protein are indispensable to rolBTR protein activity . The characteristic tyrosine phosphatase super family motif CX5R is absent in the rolBTR protein . For rolB this motif is possibly functionally relevant . We conclude that the rolBTR gene product has morphogenic activity but is not a functional homologue of the rolB protein.

Biochim Biophys Acta, 1998 Mar 4, 1396(1), 1 - 7
Novel structural difference between nopaline- and octopine-type trbJ genes: construction of genetic and physical map and sequencing of trb/traI and rep gene clusters of a new Ti plasmid pTi-SAKURA; Suzuki K et al.; We constructed a gene library of a nopaline-type Ti plasmid (called pTi-SAKURA) which was newly isolated from Agrobacterium tumefaciens MAFF301001 of a Japanese cherry tree . The partial sequencing data, which were distributed over the entire plasmid genome, made it possible to assign typical Ti-encoded genes including trb and rep gene clusters . The trb/traI and rep gene clusters were sequenced completely . All the genes in the regions except trbJ were homologous with the corresponding genes on octopine-type Ti plasmids, based on both ORF size and sequence similarity . The trbJ on pTi-SAKURA is similar to that of an octopine-type Ti, but has an extra 282-base segment in its central domain . The above gene organization and sequences suggest a divergence of Ti plasmid during evolution in relation to Rhizobium plasmids, and is discussed in this paper.

Eur J Biochem, 1998 Mar 1, 252(2), 229 - 36
Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay; Fraser PD et al.; An in vitro assay procedure for the carotenoid (beta-ionone ring) 3,3'-hydroxylase and 4,4'-oxygenase has been developed that enables efficient conversion of non-radiolabeled carotenoid substrates added directly into aqueous solution . The following enzymic conversions were demonstrated and apparent kinetic constants (Vmax, Km, and specificity constants) obtained: (a) 3,3'-hydroxylase (from Agrobacterium aurantiacum and Alcaligenes sp . strain PC-1) converted phoenicoxanthin (adonirubin) to astaxanthin, 3-hydroxyechinenone to 4-ketozeaxanthin (adonixanthin), 3'-hydroxyechinenone to 4-ketozeaxanthin, as well as echinenone to 4-ketozeaxanthin via 3- and 3'-hydroxyechinenone; (b) 4,4'-Oxygenase (from A . aurantiacum, Alcaligenes sp . strain PC-1 and Haematococcus pluvialis) converted 4-ketozeaxanthin to astaxanthin, 3-hydroxyechinenone to phoenicoxanthin, 3'-hydroxyechinenone to phoenicoxanthin, and echinenone to canthaxanthin . Determination of substrate specifities allowed assessment of biosynthetic routes to astaxanthin formation and demonstrated that pathways via mono-hydroxylated and ketolated products are enzymically feasible.

Plant Mol Biol, 1998 Apr, 36(6), 897 - 907
Characterisation and promoter analysis of the Arabidopsis gene encoding high-mobility-group protein HMG-I/Y; Gupta R et al.; The single-copy gene encoding the Arabidopsis HMG-I/Y protein was isolated and characterised . The gene encodes a protein of 204 amino acid residues and contains a single intron of 73 bp . Primer extension analysis indicates that transcription starts 115 bp upstream of the translation start and the leader sequence contains a short open reading frame of 13 amino acid residues . The 5'-upstream region of 2117 bp and several 5' deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred to tobacco by Agrobacterium-mediated transformation . Analysis of transgenic tobacco plants containing HMG-I/Y promoter regions of -2117, -1468 and -707 from the translation start detected GUS activity in all organs examined, including roots, stems, leaves and floral organs . Deletion from -707 to -185 resulted in a 20-30-fold reduction in GUS activity in roots and stems, indicating the presence of important quantitative regulatory elements in this region.

J Biotechnol, 1997 Jan 3, 59(3), 169 - 81
Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts; Misawa N et al.; The crt gene clusters responsible for the biosynthesis of carotenoids such as lycopene, beta-carotene and astaxanthin have been isolated from carotenogenic bacteria such as Erwinia species and the marine bacterium Agrobacterium aurantiacum . The functions of the individual genes have been identified . The first substrate of the enzymes encoded by the Erwinia crt clusters is farnesyl pyrophosphate which is not only the precursor for carotenoid biosynthesis but also sterols, dolichols and other numerous isoprenoid compounds . Escherichia coli does not naturally synthesize carotenoids, but by using the carotenogenic genes recombinant strains accumulating lycopene, beta-carotene and astaxanthin have been produced . Other non-carotenogenic bacteria such as Zymomonas mobilis have also been engineered to produce beta-carotene by the introduction of the corresponding crt genes . A gene capable of enhancing carotenoid levels in E . coli has also been isolated from cDNA libraries of the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis . This gene has been found to encode an isopentenyl pyrophophate isomerase . It has further been shown that the edible yeasts Candida utilis as well as Saccharomyces cerevisiae, which possess no carotenoid biosynthetic pathway, acquire the ability to produce carotenoids, when the carotenogenic genes are expressed under the control of yeast-derived promoters and terminators . It has been observed in the yeasts S . cerevisiae and C . utilis carrying the lycopene biosynthesis genes that ergosterol content is decreased by 10 and 35%, respectively . It is therefore likely that the carbon flux for the ergosterol biosynthesis has been partially directed from farnesyl pyrophosphate to a new pathway for the lycopene biosynthesis . Further, the expression of a truncated gene which codes for the catalytic domain of the endogenous 3-hydroxy-3-methylglutaryl coenzyme . A reductase, has been found to be effective for enhancing carotenoid levels in the yeast C . utilis.

Plant Cell Physiol, 1998 Jan, 39(1), 57 - 63
Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants; Okamoto M et al.; To enhance the disease resistance of plants expressing a foreign peptide, the gene for sarcotoxin IA, which is an antimicrobial peptide from an insect consisting of 39 amino acid residues, was introduced into tobacco (Nicotiana tabacum) under the control of a high expression promotor via Agrobacterium-mediated transformation . In transgenic plants, sarcotoxin IA mRNA accumulated to detectable levels, however, the amount of the peptide produced was so small that we could scarcely detect it by protein gel blot analysis, probably because of the instability of short peptides in plant cells . To improve the expression efficiency, genes for four types of amino-terminal and carboxyl-terminal translational fusions of sarcotoxin IA together with the GUS gene were introduced into tobacco . In all four types of transgenic tobacco plants, high level transcripts similar to that in the direct expression sarcotoxin IA construct were found . Protein gel blot analysis with both anti-sarcotoxin IA and GUS antibodies showed production of high levels of fusion protein in all transgenic plants . Among them, three types had abnormal membranes and phenotypes, although no such abnormalities were found in transgenic plants in which only sarcotoxin IA was expressed in a secretable form . All together, these results indicated that, for stable and effective expression of a foreign short peptide in transgenic plants, expression as a fusion protein is useful and that secretion of sarcotoxin IA outside of cells is necessary for generation of useful antimicrobial transgenic plants.

Mol Cells, 1997 Dec 31, 7(6), 783 - 7
Establishment of a transgenic tobacco cell suspension culture system for producing murine granulocyte-macrophage colony stimulating factor; Lee JS et al.; We tested if murine granulocyte-macrophage colony stimulating factor (mGM-CSF) is produced as a biologically active form through plant cell culture . The mGM-CSF gene was cloned into the plant expression vector, pBI121, and Ti-plasmid mediated transformation of tobacco leaves was conducted using Agrobacterium tumefaciens harboring the recombinant mGM-CSF (rmGM-CSF) gene . Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plants . Northern blot analysis indicated the expression of the introduced mGM-CSF gene in both transgenic plant and cell suspension cultures . In addition, the biological activity of rmGM-CSF from plant cell culture was confirmed by measuring the proliferation of GM-CSF dependent FDC-P1 cells.

Curr Microbiol, 1998 Feb, 36(2), 96 - 101
Characteristics and DNA-sequence of a cryptic haloalkanoic acid dehalogenase from Agrobacterium tumefaciens RS5; Kohler R et al.; Agrobacterium tumefaciens RS5 harbors two different hydrolytic haloalkanoic acid dehalogenase genes, one coding for a nonstereospecific enzyme (DhlS5II) and a second for a cryptic L-isomer-specific dehalogenase (DhlS5I) . The latter gene was cloned and expressed in Escherichia coli . Biochemical characterization and sequence analysis of dhlS5I shows its membership to the class of the L-isomer-specific hydrolytic dehalogenases . Highest homology of 72% was found to the dehalogenase LdexYL from Pseudomonas sp . YL . Both enzymes share an unusual high temperature optimum of 65 degrees C . Controlled by a vector promoter, high specific dehalogenase activities up to 32 U mg-1 protein were obtained in E . coli, and putatively, owing to its own sigma70-dependent promoter, a constitutive low-level expression of dhlS5I of 0.4 U mg-1 protein was measured.

FEMS Microbiol Lett, 1998 Feb 1, 159(1), 41 - 6
Overproduction of beta-glucosidase in active form by an Escherichia coli system coexpressing the chaperonin GroEL/ES; Machida S et al.; beta-Glucosidase from Cellvibrio gilvus was successfully overproduced in soluble form in Escherichia coli, with the coexpression of GroEL/ES . Without the GroEL/ES protein, the beta-glucosidase overexpressed in E . coli constituted a huge amount (80%) of the total cellular protein, but was localized in the insoluble fraction, and little activity was detected in the soluble fraction . Coexpression of the E . coli GroEL/ES had a drastic impact on the proper folding of the beta-glucosidase; 20% of the overexpressed enzyme was recovered in the soluble fraction in active form . In addition, the synergistic effect of GroEL/ES and the low induction temperature led to 70% solubilization of the total expressed target protein and more than a 20-fold increase in activity . Similar effects of GroEL/ES were also observed on the overexpressed beta-glucosidase from Agrobacterium tumefaciens.

Mol Microbiol, 1998 Jan, 27(2), 405 - 14
Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins; Das A et al.; Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins . To study the topology of the Agrobacterium T-DNA transfer proteins, we constructed a transposon, Tn3phoA . The transposon mobilizes into plasmids at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein topology directly in the bacterium of interest . For studies on the DNA transfer proteins, an Agrobacterium strain deficient in phoA under our experimental conditions was constructed by chemical mutagenesis . A plasmid containing virB and virD4 was used as a target for mutagenesis . Twenty-eight unique phoA-positive clones that mapped to eight virB genes were isolated . Multiple insertions throughout VirB1, VirB5, VirB7, VirB9 and VirB10 indicated that these proteins primarily face the periplasm . Insertions in VirB2, VirB6 and VirB8 allowed the identification of their periplasmic domains . No insertions were found in VirB3, VirB4 and VirB11 . These proteins either lack or have a short periplasmic domain . No insertions mapped to VirD4 either . To study VirD4 topology, targeted phoA fusions and random lacZ fusions were constructed . Analysis of the fusion proteins indicated that VirD4 contains a single periplasmic domain near the N-terminus, and most of the protein lies in the cytoplasm . A hypothetical model for the T-DNA transport pore is presented.

Mol Microbiol, 1998 Jan, 27(2), 289 - 97
Activity of the quorum-sensing regulator TraR of Agrobacterium tumefaciens is inhibited by a truncated, dominant defective TraR-like protein; Zhu J et al.; Horizontal transfer of Agrobacterium tumefaciens tumour-inducing plasmids requires opines, which are released from plant tumours as nutrients for the bacteria . The opine octopine causes synthesis of the quorum-sensing TraR protein, which activates several tra promoters in the presence of a pheromone called Agrobacterium autoinducer (AAI) . A gene, traS, was previously found on the same Ti plasmid in an operon that directs the uptake of mannopine, another opine . TraS strongly resembles TraR but lacks a DNA-binding module . TraS did not activate a TraR-dependent promoter and blocked TraR function, probably by forming inactive heteromultimers . Expression of traS was induced by mannopine, although this induction was strongly inhibited by the favoured catabolites succinate, glutamine and tryptone . Mannopine inhibited conjugation in a TraS-dependent fashion, and artificial overexpression of TraS also inhibited conjugation . Favoured catabolites restored tra gene expression in wild-type strains but not in strains that overexpress TraS . Downstream of traS is a gene encoding a truncated, defective chemoreceptor whose expression abolished chemotaxis.

Mol Microbiol, 1998 Jan, 27(2), 277 - 88
Octopine-type Ti plasmids code for a mannopine-inducible dominant-negative allele of traR, the quorum-sensing activator that regulates Ti plasmid conjugal transfer; Oger P et al.; Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by two hierarchical signalling systems . Transfer is dependent on a subset of opines produced by the plant tumours induced by the bacterium . Induction also requires an acyl-homoserine lactone signal, called AAI, that is produced by the bacteria themselves . AAI is the co-inducer for TraR, the transcriptional activator required for expression of the tra regulon . Octopine induces conjugation of the octopine-mannityl opine-type Ti plasmids by regulating the expression of traR via OccR, the octopine-dependent activator of the opine regulon . We have discovered a second traR-like gene, trlR, on the octopine-mannityl opine-type Ti plasmids pTi15955 and pTiR10 . This gene is located in an operon coding for a mannopine transport system and is expressed as part of the mannityl opine regulon . Sequence analysis indicated that trlR is a frameshift allele of traR, and the resulting protein lacks the carboxy-terminal domain thought to constitute the DNA-binding region of TraR . Expression of trlR inhibited octopine-induced conjugation of pTi15955 and pTiR10 by suppressing the TraR-mediated transcription of the tra and trb operons . Although TrlR had no effect on the expression of traR, TraR activated the expression of trlR . Southern hybridizations indicated that several other Ti and opine-catabolic plasmids contain more than one copy of genes homologous to traR . We propose that trlR is a dominant negative allele of traR and that TrlR inhibits conjugation by forming inactive heteromultimers with TraR.

Plant Mol Biol, 1998 Feb, 36(3), 387 - 92
Excision of Ds1 from the genome of maize streak virus in response to different transposase-encoding genes; Shen WH et al.; We have previously established a reverse genetic system for studying excision of the transposable element Ds1 in maize plants . Ds1 carried by the genome of maize streak virus (MSV) is introduced into maize plants by agroinfection . Excision of Ds1 from the MSV genome depends on the presence of an active Ac element in the recipient maize plants . With the purpose of exploiting MSV-Ds1 as vector for maize transformation, we studied different genes encoding the transposase (TPase) for their efficiency of activating Ds1 excision . These genes were inserted in the same T-DNA carrying MSV-Ds1 and introduced into maize plants by Agrobacterium-mediated transformation . We showed that the wild-type TPase transcribed by the 2' promoter produced much higher efficiency of Ds1 excision than that transcribed by the Ac promoter . In contrast to what had been observed in tobacco and petunia, the truncated TPase (103-807) lacking the amino-terminal 102 amino acids gave a much more reduced Ds1 excision efficiency than the wild-type TPase when both genes were transcribed by the 2' promoter.

Plant Mol Biol, 1998 Jan, 36(1), 89 - 99
Structural analysis of the nit2/nit1/nit3 gene cluster encoding nitrilases, enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana; Hillebrand H et al.; A 13.8 kb DNA sequence containing the promoters and the structural genes of the Arabidopsis thaliana nit2/nit1/nit3 gene cluster has been isolated and characterized . The coding regions of nit2, nit1 and nit3 spanned 1.9, 1.8 and 2.1 kb, respectively . The architecture of the three genes is highly conserved . Each isoform consists of five exons separated by four introns . The introns are very similar with respect to size and position, but differ considerably in sequence composition . In contrast to the coding sequences the three promoters are very different in sequence, size and in their repertoire of cis elements, suggesting differential regulation of the three nitrilase isoenzymes by the developmental program of the plant and by diverse environmental factors . The nit1 promoter was subjected to analysis in planta . Translational fusions placing the nit1 full-length promoter and a series of 5'-deletion fragments in front of the uidA gene encoding beta-glucuronidase (GUS) were used for Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum . GUS expression was highest in fully expanded leaves and in the shoot apex as well as in the apices of developing lateral buds, whereas the GUS activity displayed by developing younger leaflets was restricted to the tips of the expanding leaves . Within the root tissue GUS expression was restricted to the root tips and the tips of newly forming lateral roots . Structural features of the nitrilase gene family and nitrilase gene expression patterns are discussed in context with current knowledge of auxin biosynthesis and auxin effects on different tissues.

Plant Mol Biol, 1998 Jan, 36(2), 205 - 17
Analysis of T-DNA-mediated translational beta-glucuronidase gene fusions; Kertbundit S et al.; Three random translational beta-glucuronidase (gus) gene fusions were previously obtained in Arabidopsis thaliana, using Agrobacterium-mediated transfer of a gus coding sequence without promoter and ATG initiation site . These were analysed by IPCR amplification of the sequence upstream of gus and nucleotide sequence analysis . In one instance, the gus sequence was fused, in inverse orientation, to the nos promoter sequence of a truncated tandem T-DNA copy and translated from a spurious ATG in this sequence . In the second transgenic line, the gus gene was fused to A . thaliana DNA, 27 bp downstream an ATG . In this line, a large deletion occurred at the target site of the T-DNA . In the third line, gus is fused in frame to a plant DNA sequence after the eighth codon of an open reading frame encoding a protein of 619 amino acids . This protein has significant homology with animal and plant (receptor) serine/threonine protein kinases . The twelve subdomains essential for kinase activity are conserved . The presence of a potential signal peptide and a membrane-spanning domain suggests that it may be a receptor kinase . These data confirm that plant genes can be tagged as functional translational gene fusions.

Can J Microbiol, 1997 Dec, 43(12), 1164 - 71
EcbI and EcbR: homologs of LuxI and LuxR affecting antibiotic and exoenzyme production by Erwinia carotovora subsp . betavasculorum; Costa JM et al.; Erwinia carotovora subsp . betavasculorum Ecb168 causes vascular necrosis and root rot of sugar beet and produces an antibiotic(s) that is antagonistic against other Erwinia spp . EcbI- mutants of Ecb168, each containing a single transposon insertion in the ecbI gene (for Erwinia carotovora subsp . betavasculorum inducer), do not produce detectable levels of extracellular protease or antibiotic(s), and express less pectate lyase activity and virulence than the wild-type strain . A plasmid containing the cloned ecbI gene complemented the EcbI- mutants for these phenotypes . Protease production by EcbI- mutants grown on agar surfaces was restored by neighboring cells of Escherichia coli containing ecbI . Production of a diffusible N-acylhomoserine lactone autoinducer by wild-type Ecb168 was detected with indicator strains of E . coli and Agrobacterium tumefaciens . EcbI- mutant strains did not produce an autoinducer detected by the indicator strains . Antibiotic production by EcbI- mutants was restored by cell-free culture supernatants of Ecb168 or E . coli containing a cloned ecbI gene . The predicted amino acid sequence of EcbI is similar to those of CarI, ExpI, and HsII, three LuxI homologs required for production of a diffusible N-acylhomoserine lactone autoinducer in Erwinia carotovora subsp . carotovora . A luxR homolog, termed ecbR (for Erwinia carotovora subsp . betavasculorum regulator), is convergently transcribed and overlaps with ecbI by 17 bp at their 3' ends . These results are consistent with the hypothesis that a quorum-sensing system related to the prototypic luxI-luxR gene pair controls antibiotic and exoenzyme production in Erwinia carotovora subsp . betavasculorum.

Plasmid, 1998, 39(1), 35 - 40
Incompatibility properties of tartrate utilization plasmids derived from Agrobacterium vitis strains; Szegedi E et al.; The incompatibility properties of two tumor-inducing (Ti) and seven tartrate (Tr) plasmids, derived from various Agrobacterium vitis strains, were characterized using incRh1, incRh2, incRh3, and incRh4 clones which were established for the identification and classification of Agrobacterium plasmids . The tested A . vitis plasmids could be allocated into four groups on the basis of their incompatibility with incRh1, incRh2, and incRh4 clones . The two octopine tumor-inducing plasmids, pTiAT6 and pTiAB3, expressed incompatibility both to incRh1 and to incRh2 clones . Three pTrs could not be allocated either to incRh1-4 and incAg1 or to the wide-host-range incP1, incQ, and incW groups.

J Biotechnol, 1997 Dec 3, 58(3), 147 - 56
Tanshinone production in Ti-transformed Salvia miltiorrhiza cell suspension cultures; Chen H et al.; Transformed cell cultures of Salvia miltiorrhiza were established by infecting sterile plantlets with Agrobacterium tumefaciens strain C58 . The transformed cells in suspension formed macroscopic clumps of cell aggregates up to 2-3 cm in size rather than homogeneous cell suspensions . These transformed cells grew well in hormone-free media . It was found that the B5 mediums supported the best growth while the 6,7-V medium promoted tanshinone production in the transformed cell suspension cultures . The effect of initial sucrose concentration on cell growth was also studied . The best growth was observed when cells were cultivated in the B5 medium containing 30 g l-1 sucrose . Although low levels of tanshinones were produced in fast growing cell cultures, there existed a rapid increase in tanshinone production when the cell aggregates were transferred to the fresh yeast-extract-containing medium . By this two-stage culture method, about 22 mg tanshinones were produced in 1 liter of medium . Green cell aggregates were formed when cells were cultured under illumination . Light was found to have an inhibitory effect on tanshinone biosynthesis . A sensitive high-performance liquid chromatographic method was developed for the measurement of tanshinones . Cryptotanshinone, tanshinone I and tanshinone IIA were identified from the transformed cultures.

Plant J, 1997 Oct, 12(4), 945 - 8
High-efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T-DNA 1' promoter; Mengiste T et al.; Two selectable marker genes harbouring the bar coding region but differing in their promoters were compared in an Arabidopsis thaliana transformation assay using in planta infiltration with Agrobacterium tumefaciens . Surprisingly, in four Arabidopsis ecotypes examined, the 1' promoter from the right T-DNA was superior to the most commonly used 35S promoter of cauliflower mosaic virus (CaMV) . The ecotype Wassilewskija gave the highest transformation frequencies, with an average of between 5.3 and 6.3% of the seedlings subjected to the selection . This is approximately 30-fold higher than previously reported results . Analysis of T-DNA integration patterns in single transformed plants or pooled populations revealed independent T-DNA integration events in each case . Results show that the 1' promoter is an attractive alternative to the 35S promoter for the generation of T-DNA insertion lines . The 1' promoter may be especially beneficial for the secondary transformation of transgenic strains containing the 35S promoter to exclude homology-mediated gene silencing.

Plant J, 1997 Oct, 12(4), 937 - 43
Internal AU-rich elements modulate activity of two competing 3' splice sites in plant nuclei; Merritt H et al.; In vivo analyses using an autonomously replicating Agrobacterium/geminivirus vector have enabled identification of AU-rich intronic elements critical for 5' and 3' splice site selection in dicot plant nuclei and development of a model for pre-mRNA intron recognition in plant nuclei . To determine the minimal length, spacing and nucleotide compositions constraining recognition of the 3' boundary of an intron, two or four nucleotide substitutions have been introduced into the two AU-rich elements located between 50 and 66 nucleotides upstream from the 3' splice site of maize Adh1 intron 3 . In each case tested, substitutions in the distal left element (-62 to -66) inactivate the downstream 3' splice site at -1 more effectively than substitutions in the proximal right element (-50 to -55) . Guanosine or cytosine substitutions in either element reduce recognition of the -1 site significantly; adenosine substitutions have a less severe effect . Mutations in both of these AU elements additively block recognition of the downstream 3' splice site . The strong additive effect of these mutations supports a model in which short sets of AU islands bind interactive factors and cooperatively modulate usage of the downstream splice site . In contrast to the uridine requirements documented for the 3' terminus of plant introns, adenosines are partially interchangeable with uridines within this internal region of the intron.

Plant Mol Biol, 1997 Nov, 35(5), 633 - 40
Expression of glyoxylate cycle genes in cucumber roots responds to sugar supply and can be activated by shading or defoliation of the shoot; Ismail I et al.; When cucumber roots are excised and incubated without a carbon source, isocitrate lyase (ICL) and malate synthase (MS) mRNAs increase significantly in amount . However, if sucrose is added to the excised roots, the mRNAs do not accumulate . Hairy roots obtained by transformation with Agrobacterium rhizogenes show the same response . Transgenic hairy roots containing the Icl and Ms gene promoters fused to the GUS reporter gene, have very low GUS activity which increases dramatically when roots are incubated in the absence of sugar, indicating regulation at the transcriptional level . Staining of sugar-deprived roots shows that GUS activity is concentrated mainly in root tips and lateral root primordia, where demand for carbohydrate is greatest . In order to determine if Icl and Ms genes are expressed in roots of whole plants under conditions which may occur in nature, cucumber plants were subjected to reduced light intensity or defoliation . In both cases increases were observed in ICL and MS mRNAs . These treatments also reduced root sugar content, consistent with the hypothesis that sugar supply could control expression of Icl and Ms genes in roots of whole plants.

Plant Mol Biol, 1997 Nov, 35(4), 523 - 30
Gene targeting approaches using positive-negative selection and large flanking regions; Thykjaer T et al.; We report here on strategies aimed at improving the frequency of detectable recombination in plants by increasing the efficiency of selecting double-recombinants in transgenic calli . Gene targeting was approached on the Gln1 and the Pzfloci of Lotus japonicus, using Agrobacterium tumefaciens T-DNA replacement vectors . Large flanking regions, up to 22.9 kb, surrounding a positive selection marker were presented as substrates for homologous recombination . For easier detection of putative recombinants the negative selectable marker cytosine deaminase was inserted at the outside borders of the flanking regions offered for cross-over . A combination of positive and negative selection allowing double-recombinants to grow, while counter-selecting random insertions, was used to select putative targeting events . The more than 1000-fold enrichment observed with replacement vectors designed to minimize gene silencing demonstrated the efficiency of the negative selection . Using five different replacement vectors an estimated total of 18,974 transformation events were taken through the positive-negative selection procedure and 185 resistant calli obtained . Targeting events could not be verified in the survivors by PCR screening and Southern blot analysis . With this approach the frequency of detectable gene targeting in L . japonicus was below 5.3 x 10(-5), despite the large flanking sequences offered for recombination.

Plant Mol Biol, 1997 Nov, 35(4), 509 - 22
Differential modification of flavonoid and isoflavonoid biosynthesis with an antisense chalcone synthase construct in transgenic Lotus corniculatus; Colliver SP et al.; Three clonal genotypes of Lotus corniculatus L . (bird's foot trefoil) were transformed with an antisense chalcone synthase (CHS) gene construct made using a stress induced CHS17 cDNA from Phaseolus vulgaris under the control of the constitutive CaMV 35S promoter and Nos terminator via Agrobacterium rhizogenes . After initial screening, ten antisense and five control co-transformation events from each recipient clonal genotype were analysed . After elicitation with glutathione, the level of tannin accumulation was found to be increased in a number of antisense root cultures derived from the low (S33) and moderate (S50) tannin recipient genotypes . Six antisense and four control transformed lines from genotype S50 were selected for more detailed study . The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from 1 to 5 and antisense orientation was confirmed by PCR . In transformed root cultures, increased CHS transcript levels were noted in a number of antisense lines . Biochemical analyses of glutathione-elicited-root cultures indicated a significant increase in tannin accumulation in antisense CHS lines and mean vestitol levels were reduced . These results show that the introduction of a heterologous antisense chalcone synthase construct into L . corniculatus resulted in an unpredicted molecular and biochemical phenotype . Such findings are discussed in relation to manipulation of this complex multigene family.

Plant Mol Biol, 1997 Nov, 35(4), 425 - 31
A promoter directing high level expression in pistils of transgenic plants; Ficker M et al.; The promoter of the potato (Solanum tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase, was cloned . Various fragments of the SK2-promoter, from 1 kb down to 0.23 kb in length, were fused to the GUS reporter gene . Chimaeric SK2 promoter-GUS fusion constructs were transformed into potato by Agrobacterium tumefaciens-mediated transformation . The SK2-GUS transgenic potato plants exhibited a highly specific GUS activity in the pistil . Expression in the pistil was shown to be developmentally regulated . In addition to the GUS activity in pistils, transgenic plants also showed a much weaker ectopic expression in anthers . In other tissues no systematic expression was detectable . All SK2 promoter fragments analysed conferred pistil-specific expression without significant qualitative or quantitative differences, demonstrating that the regulatory elements mediating this expression pattern are located within a 230 bp SK2 promoter fragment . The SK2 promoter may be used to engineer high levels of expression in pistils of transgenic plants.

Plant Mol Biol, 1997 Oct, 35(3), 323 - 30
Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures; Wallis JG et al.; A synthetic antifreeze protein gene was expressed in plants and reduced electrolyte leakage from the leaves at freezing temperatures . The synthetic AFP was expressed as a fusion to a signal peptide, directing it to the extracytoplasmic space where ice crystallization first occurs . The gene was introduced to Solanum tuberosum L . cv . Russet Burbank by Agrobacterium-mediated transformation . Transformants were identified by PCR screening and expression of the introduced protein was verified by immunoblot . Electrolyte-release analysis of transgenic plant leaves established a correlation between the level of transgenic protein expression and degree of tolerance to freezing . This is the first identification of a phenotype associated with antifreeze protein expression in plant tissue.

Mol Plant Microbe Interact, 1998 Feb, 11(2), 131 - 43
Opine catabolic loci from Agrobacterium plasmids confer chemotaxis to their cognate substrates; Kim H et al.; Opines are carbon compounds produced by crown galls and hairy roots induced by Agrobacterium tumefaciens and A . rhizogenes, respectively . These novel condensation products of plant metabolic intermediates are utilized as nutritional sources by the Agrobacterium strains that induced the growths . Thus, opines are thought to favor the propagation of agrobacteria in the tumorsphere . Certain Agrobacterium strains were chemoattracted to opines . The chemotactic activities to octopine, to nopaline, to mannopine, and to agrocinopines A + B were dependent on the type of the Ti plasmid present in the bacterium . The determinants for chemotaxis to these opines were localized to the regions of the octopine- and nopaline-type Ti plasmids coding for transport and catabolism of that opine . An insertion in accA, which encodes the periplasmic binding protein for agrocinopines A + B, abolished chemotaxis while an insertion in accC, which encodes a component of the transport system, and an insertion in accF, which encodes a function required for agrocinopine catabolism, did not affect chemotaxis to this opine . Thus, transport and catabolism of these opines are not required for the chemotactic activity . Analyses of subclones of the acc region confirmed that accA is the only gene required from the Ti plasmid for chemotaxis to agrocinopines A + B.

Planta, 1998 Jan, 204(1), 70 - 7
Evidence of protein-tyrosine kinase activity in Catharanthus roseus roots transformed by Agrobacterium rhizogenes; Rodriguez-Zapata LC et al.; Homogenate fractions (soluble and particulate) from transformed roots of Catharanthus roseus (L.) G . Don showed several phosphorylated proteins when incubated with gamma-{32P}ATP . The phosphorylation in the proteins of 55, 40, 25, 18 and 10 kDa in the particulate fraction and 63 kDa in the soluble fraction was resistant to alkali treatment . Several proteins in both fractions gave a positive signal with monoclonal antiphosphotyrosine antibodies . In-situ phosphorylation in both fractions showed several proteins that cross-reacted with the antiphosphotyrosine antibodies . Tyrosine kinase activity was detected using an exogenous substrate RR-SRC, a synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src . This activity was inhibited by genistein, a tyrosine kinase inhibitor . These results indicate, for the first time, the presence of protein-tyrosine kinase (EC 2.7.1.112) activity in transformed plant tissues.

J Bacteriol, 1998 Jan, 180(2), 430 - 4
Role of Agrobacterium virB genes in transfer of T complexes and RSF1010; Fullner KJ; Nonpolar virB mutants of Agrobacterium tumefaciens were tested for RSF1010 mobilization and extracellular complementation . virB2 to virB11 were essential for transfer in both assays . virB1 was essential only for high frequency transfer of RSF1010 and VirE2 . Coordinated transfer of a preassembled T complex is supported by these data and competition studies.

Mol Biotechnol, 1997 Dec, 8(3), 223 - 31
Agrobacterium-mediated transformation of élite indica and japonica rice cultivars; Zhang J et al.; A rapid, efficient, routine system has been established for Agrobacterium tumefaciens-mediated production of hundreds of fertile transgenic plants from commercially important rice cultivars, including an indica cultivar, Pusa Basmati 1 . Calli induced from embryos of mature rice seeds were cocultivated with A . tumefaciens strain LBA4404 carrying the plasmid pTOK233, then exposed to hygromycin selection followed by an efficient regeneration system . Based on the total number of calli co-cultivated, the transformation frequencies of independent transgenic rice plants including cultivars Pusa Basmati 1, E-yi 105, E-wan 5 and Zhong-shu-wan-geng, were 13.5, 13.0, 9.1, and 9.3%, respectively . T1 seeds were harvested within 7-8 mo of initiation of mature embryo cultures . Data from Southern hybridization analysis proved that foreign genes on T-DNA were stably integrated into the rice genome at low copy/site numbers . Mendelian inheritance of the transgenes was confirmed in T1 progeny.

Mol Biotechnol, 1997 Dec, 8(3), 199 - 213
pBECKS . A flexible series of binary vectors for Agrobacterium-mediated plant transformation; McCormac AC et al.; A series of binary T-DNA vectors (pBECKS) has been created for use in the Agrobacterium-mediated genetic transformation of plants . The pBECKS series has corrected the undesirable features of the popular pBIN19 vector; the deleterious mutation within the coding sequence of nptII has been amended and the cloning sites are now adjacent to the right border repeat in order to reduce the possibility of producing truncated sequences of novel genes within transformants . One set of vectors incorporates various combinations of the marker genes gusA, C1/Lc, nptII, hph, and bar, for pursuit of early and stable transformation events . A set of constructs which contain deleted T-DNA borders in various combinations and display predictably altered efficacies for gene transfer has also been created . A modular set of vectors has been designed to facilitate the insertion and transfer of novel gene sequences by providing a nptII-linked plant expression cassette or lacZ-multiple cloning site . A range of antibiotic resistance genes has been incorporated into the non-T-DNA part of the vectors in order to facilitate their selection across the range of Agrobacterium virulence strains.

Plant Cell, 1997 Dec, 9(12), 2171 - 81
Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo; Forreiter C et al.; Using Agrobacterium, we developed a method to transform an Arabidopsis cell suspension culture . A stably transformed cell line expressing high levels of firefly luciferase (Luc) was used for in vivo studies of thermal denaturation and renaturation of the enzyme and the protective role of different chaperones . Luc activity was monitored under heat stress and recovery conditions in control, thermotolerant cells and cells expressing plant chaperones after transient cotransformation with plasmids encoding proteins of the heat shock protein Hsp90, Hsp70, or Hsp20 family . The effects of the expressed proteins were specific . The Hsp17.6 class I protein maintained Luc activity on a level comparable with that observed in thermotolerant cells and improved Luc renaturation . Although transient expression of Hsp90 did not protect Luc from thermal denaturation, it accelerated Luc renaturation during recovery . In contrast to the other chaperones tested, overexpression of Hsp70 alone had no effect on denaturation and renaturation of Luc but enhanced Luc renaturation if coexpressed with Hsp17.6.

Chin J Biotechnol, 1997, 13(3), 207 - 10
Selection of a high tanshinone-producing crown gall strain and production of tanshinone in the strain; Song J et al.; Crown galls were induced by direct infection of sterile seedlings of Salvia miltiorrhiza with Agrobacterium tumefaciens C58 and subcultured on a 67-V hormone-free medium by successively selecting red cell aggregates . A high tanshinone-producing crown gall strain C1 was obtained after 12 months in a subculture . It grows well and retains its high tanshinone-producing characteristic in liquid stationary cultures . It is obvious that the yeast extract and fermentation extract of Armillaria mellea as elicitors promote strain C1 to produce tanshinone . Tanshinone content of strain C1 cultures was over three times higher than that of the crude drugs . The results indicated that the crown gall tissue and elicitor technique could provide some new clues for the production of tanshinone under the condition of a liquid stationary culture.

Chin J Biotechnol, 1997, 13(3), 153 - 9
Expression of the spinach betaine aldehyde dehydrogenase (BADH) gene in transgenic tobacco plants; Liang Z et al.; Plasmid pLS9 contains a 1.5-kb of spinach cDNA including its complete open reading frame . The 1.5-kb BADH cDNA was cut from pLS9 using restriction enzyme and was inserted into the expression cassette of plasmid pYH between the CaMV 35S promoter and polyA signal sequence . The 35S-BADH cDNA-polyA fragment of pYH was cloned into a polylinker cloning site of the binary vector pBin19 . The resulting plasmid pBinBADH-S was transferred to Agrobacterium tumefacies LBA4404 . The tobacco plants were transformed with strain LBA4404 containing pBinBADH-S, and more than ninety kanamycin-resistant transformants were selected . Polymerase chain reaction (PCR) detection showed that more than 60% of the transformed tobacco plants contained the foreign BADH gene . The Western blot analysis, BADH enzymatic assay, specific stain for BADH activity, and the test for salt tolerance showed that BADH gene was normally expressed in the transgenic tobacco plants . The BADH enzymes also presented in chloroplasts and cytosol of the transgenic plants . The transgenic tobacco plants having strong expression of BADH gene had strong ability to tolerate high salt stress.

Acta Microbiol Pol, 1997, 46(2), 145 - 56
A new PCR system for Agrobacterium tumefaciens detection based on amplification of T-DNA fragment; Sachadyn P et al.; The design of the PCR system presented in this work is based on the knowledge of the molecular character of the crown gall disease . The virulence of Agrobacterium tumefaciens requires the presence of a big (up to 235,000 bp) plasmid Ti (pTi-tumour inducing plasmid) . This plasmid carries the so-called T-DNA fragment (T-DNA-transferred DNA), which integrates into cell chromosomes of the infected plants and subsequently changes the plant morphology nad metabolism . In cannot be excluded that after T-DNA integration the presence of Agrobacterium is not necessary for the development of pathological changes . Thus, T-DNA is the only sign that must be present both in virulent bacteria and in infected plants in any stadium of the disease and even before the infection . This is why T-DNA was chosen as the target region for PCR amplification . Primers flanking a 220 bp fragment of one of the conservative regions responsible for Agrobacterium pathogenicity, namely tms2 gene coding for indolacetamide amidohydrolase (the second step of auxin biosynthesis) were designed as the optimal for PCR amplification . The PCR amplification reactions were performed for matrixes isolated from cultures of reference strains giving one predicted product for each sample . First attempts of T-DNA detection in infected soils and plants were performed . We hope that the presented new PCR system for Agrobacterium tumefaciens detection will help to fight the crown gall disease in the nearest future.

Mol Plant Microbe Interact, 1998 Jan, 11(1), 57 - 63
The omega sequence of VirD2 is important but not essential for efficient transfer of T-DNA by Agrobacterium tumefaciens; Bravo-Angel AM et al.; The VirD2 protein of Agrobacterium tumefaciens contains defined sequences necessary for processing and transferring the T-DNA during transformation of plant cells . We performed a mutational analysis of the conserved omega sequence of VirD2, whose role has proven to be difficult to elucidate so far . In this report, we show that a deletion of these 5 amino acids or their replacement by 5 glycines reduced T-DNA transfer considerably, compared with wild type, demonstrating that the omega sequence is important for the efficient transfer of T-DNAs . However, the efficiency and pattern of integration of the T-DNAs were not affected by any modifications of the omega sequence . The importance of the C terminus of VirD2 for T-DNA transfer is discussed.

Transgenic Res, 1997 Nov, 6(6), 403 - 13
Expression of cholera toxin B subunit oligomers in transgenic potato plants; Arakawa T et al.; A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter . Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated . The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification . Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (M(r) approximately 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues . Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (M(r) approximately 15 kDa) during heat or acid treatment . The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein . Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin . In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties . The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world.

J Bacteriol, 1998 Jan, 180(1), 20 - 6
Succinoglycan production by Rhizobium meliloti is regulated through the ExoS-ChvI two-component regulatory system; Cheng HP et al.; The Rhizobium meliloti exoS gene is involved in regulating the production of succinoglycan, which plays a crucial role in the establishment of the symbiosis between R . meliloti Rm1021 and its host plant, alfalfa . The exoS96::Tn5 mutation causes the upregulation of the succinoglycan biosynthetic genes, thereby resulting in the overproduction of succinoglycan . Through cloning and sequencing, we found that the exoS gene is a close homolog of the Agrobacterium tumefaciens chvG gene, which has been proposed to encode the sensor protein of the ChvG-ChvI two-component regulatory system, a member of the EnvZ-OmpR family . Further analyses revealed the existence of a newly discovered A . tumefaciens chvI homolog located just upstream of the R . meliloti exoS gene . R . meliloti ChvI may serve as the response regulator of ExoS in a two-component regulatory system . By using ExoS-specific antibodies, it was found that the ExoS protein cofractionated with membrane proteins, suggesting that it is located in the cytoplasmic membrane . By using the same antibodies, it was shown that the exoS96::Tn5 allele encodes an N-terminal truncated derivative of ExoS . The cytoplasmic histidine kinase domain of ExoS was expressed in Escherichia coli and purified, as was the R . meliloti ChvI protein . The ChvI protein autophosphorylated in the presence of acetylphosphate, and the ExoS cytoplasmic domain fragment autophosphorylated at a histidine residue in the presence of ATP . The ChvI protein was phosphorylated in the presence of ATP only when the histidine kinase domain of ExoS was also present . We propose a model for regulation of succinoglycan production by R . meliloti through the ExoS-ChvI two-component regulatory system.

Plant Physiol, 1997 Dec, 115(4), 1691 - 8
Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells; Zhu Y et al.; Reporter strains of soil-borne bacteria were used to test the hypothesis that chemicals released by root border cells can influence the expression of bacterial genes required for the establishment of plant-microbe associations . Promoters from genes known to be activated by plant factors included virE, required for Agrobacterium tumefaciens pathogenesis, and common nod genes from Rhizobium leguminosarum bv viciae and Rhizobium meliloti, required for nodulation of pea (Pisum sativum) and alfalfa (Medicago sativum), respectively . Also included was phzB, an autoinducible gene encoding the biosynthesis of antibiotics by Pseudomonas aureofaciens . The virE and nod genes were activated to different degrees, depending on the source of border cells, whereas phzB activity remained unaffected . The homologous interaction between R . leguminosarum bv viciae and its host, pea, was examined in detail . Nod gene induction by border cells was dosage dependent and responsive to environmental signals . The highest levels of gene induction by pea (but not alfalfa) border cells occurred at low temperatures, when little or no bacterial growth was detected . Detached border cells cultured in distilled water exhibited increased nod gene induction (ini) in response to signals from R . leguminosarum bv viciae.

Plant Physiol, 1997 Dec, 115(4), 1359 - 69
Sequencing, genomic organization, and preliminary promoter analysis of a black cherry (R)-(+)-mandelonitrile lyase gene; Hu Z et al.; The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10) plays a key role in cyanogenesis in rosaceous stone fruits . An MDL gene (mdl3) and its corresponding cDNA (MDL3) were isolated from black cherry (Prunus serotina) and characterized . The mdl3 gene contains 2292 bp of the 5' flanking region, the entire coding region, and 300 bp of the 3' flanking region . The coding region is interrupted by three short introns, of which one possesses the usual GC-AG splice junction dinucleotides . This gene encodes a polypeptide of 573 amino acids that includes a putative signal sequence, 13 potential N-glycosylation sites, and a presumptive flavin adenine dinucleotide-binding site . To determine whether the 5' flanking region of the mdl3 gene is capable of driving MDL expression, it was fused to the beta-glucuronidase reporter gene for Agrobacterium-mediated transformation into tobacco . Matching endogenous MDL expression patterns, beta-glucuronidase staining was observed in maturing embryos and seeds; it also occurred in postembryonic tissues, especially in association with vascular tissues . After developing a homologous transient transformation system to facilitate identification of putative regulatory sequences, we demonstrated that 125 bp (-107 to +18) of the 5' flanking sequence of the mdl3 gene is sufficient for MDL expression in protoplasts derived from immature black cherry embryos.

Mol Gen Genet, 1997 Nov, 256(5), 581 - 5
Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing; Schob H et al.; Agroinfiltration--the infiltration of Agrobacterium tumefaciens into intact plant levels--provides a rapid and simple way of screening large numbers of transgene constructs for silencing in response to a resident transgene . Transgenic Nicotiana sylvestris plants homozygous for the tobacco class I chitinase A gene CHN48 under the control of the cauliflower mosaic virus 35S RNA promoter (P35S) show a high incidence of postranscriptional gene silencing . We forced suspensions of A . tumefaciens, carrying P35S-CHN48 in a binary Tiplasmid vector, into wild-type and transgenic N, sylvestris leaves with a blunt-tipped plastic syringe . The infiltrated CHN48 transgene was expressed in leaves transformed with the vector alone, but not in CHN48-transformed leaves showing the silent phenotype . In contrast, expression of a chimeric P35S-E . coli beta-glucuronidase gene (uidA) infiltrated into leaves was not affected by the presence of the CHN48 transgene stably integrated in the host genome . These results show that extra copies of CHN48 are silenced by resident, silent copies of the same gene and confirm that CHN48 silencing is not the result of promoter interactions . The results also suggest that silencing of the additional CHN48 copies does not require their integration into chromosomes.

Gene, 1997 Nov 12, 201(1-2), 55 - 62
Pattern of DNA binding of nuclear proteins to the proximal Agrobacterium rhizogenes rolC promoter is altered during somatic embryogenesis of carrot; Fujii N; Carrot cells cultured in vitro in a medium supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) proliferate as unorganized cell clusters . Upon removal of 2,4-D from the culture medium, these cells undergo somatic embryo formation through globular, heart, and torpedo stages . Since the proximal -255 bp upstream region of the rolC gene of the Ri plasmid confers somatic embryogenesis-related activation on the uidA gene in transgenic carrot cell culture, we investigated the interaction of nuclear proteins with the proximal -255bp upstream sequences to characterize the mechanism of somatic embryogenesis-related activation . Gel retardation experiments revealed that there were several different profiles of the relative levels of DNA binding activities in nuclear protein extracts from calli, PEMs (proembryogenic masses), globular embryos, and heart/torpedo embryos . The binding activity associated with a fragment (-203 bp to -92 bp) of one protein (BI) was most abundant in globular embryos . Another DNA binding protein (AII) showed the highest DNA binding activity in calli, but had low binding activities in PEMs and globular embryos . Such an altered pattern of DNA binding activities of nuclear proteins may contribute to somatic embryogenesis-related activation of the rolC promoter . Competition experiments with oligonucleotides revealed that the BI protein interacts with AT-rich sequences.

Biochim Biophys Acta, 1997 Oct 9, 1354(1), 55 - 7
Brucella abortus arginase and ornithine cyclodeaminase genes are similar to Ti plasmid arginase and ornithine cyclodeaminase; Kim J et al.; Brucella abortus arginase and ornithine cyclodeaminase genes have been cloned and sequenced . These gene sequences are located in the same operon and occur in the same order as the homologous genes in Agrobacterium tumefaciens Ti C58 plasmid . The nucleotide sequences of the two genes have 72% and 65% identity to the respective Ti plasmid genes . Both genes are present in a single copy, and expression of arginase is regulated in response to arginine.

Gene, 1997 Oct 24, 200(1-2), 107 - 16
A binary-BAC system for plant transformation with high-molecular-weight DNA; Hamilton CM; A binary-BAC (BIBAC) vector suitable for Agrobacterium-mediated plant transformation with high-molecular-weight DNA was constructed . A BIBAC vector is based on the bacterial artificial chromosome (BAC) library vector and is also a binary vector for Agrobacterium-mediated plant transformation . The BIBAC vector has the minimal origin region of the Escherichia coli F plasmid and the minimal origin of replication of the Agrobacterium rhizogenes Ri plasmid, and thus replicates as a single-copy plasmid in both E . coli and in A . tumefaciens . The T-DNA of the BIBAC vector can be transferred into the plant nuclear genome . As examples, a 30-kb yeast genomic DNA fragment and a 150-kb human genomic DNA fragment were inserted into the BIBAC vector; these constructs were maintained in both E . coli and A . tumefaciens . In order to increase the efficiency of transfer of unusually large BIBAC T-DNAs, helper plasmids that carry additional copies of A . tumefaciens virulence genes virG and virE were constructed . These helper plasmids are compatible with, and can be present in addition to, the BIBAC vector in the A . tumefaciens host . This report details the components of the BIBAC system, providing information essential to the general understanding and the application of this new technology.

J Bacteriol, 1997 Dec, 179(24), 7796 - 802
Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter; Snape JR et al.; Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced . The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity . It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite . The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins . GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.

Mol Plant Microbe Interact, 1997 Dec, 10(9), 1087 - 93
Iron-dependent transcription of the regulatory gene ros of Agrobacterium radiobacter; Hussain H et al.; Transcription of the regulatory gene ros of Agrobacterium radiobacter requires growth in the presence of Fe although this regulation was not mediated by ros itself . The ros gene repressed its own transcription, independently of the Fe status of the growth media . It was shown that the two cysteine residues in the Ros protein were essential for the complementation of the exopolysaccharide synthesis defect of ros mutant strains . It was found that the mutation in one exo mutant strain that was complemented both by ros and by the "structural" exoY gene was not, in fact, in ros but is in some other, unknown gene . The two cysteines were also essential for the correction of this mutant . This mutant affected the expression of exoY but not of ros.

Mol Plant Microbe Interact, 1997 Dec, 10(9), 1065 - 74
Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins; Duan YP et al.; Tobacco plants were transformed with the movement protein (pathogenicity) gene (BC1) from tomato mottle geminivirus (TMoV), using Agrobacterium-mediated transformation . Different transgenic tobacco lines that expressed high levels of the BC1 protein had phenotypes ranging from plants with severe stunting and leaf mottling (resembling geminivirus symptoms) to plants with no visible symptoms . The sequence data for the BC1 transgene from the transgenic plants with the different phenotypes indicated an association of spontaneously mutated forms of the BC1 gene in the transformed tobacco with phenotype variations . One mutated transgene associated with an asymptomatic phenotype had a major deletion at the C terminus of 119 amino acid residues with a recombination resulting in the addition of 26 amino acid residues of unidentified origin . This asymptomatic, mutated BC1 attenuated the phenotypic expression of the symptomatic BC1 in a tobacco line containing both copies of the BC1 gene . Another mutated form of the BC1 gene amplified from an asymptomatic, multicopy transgenic tobacco plant did not induce symptoms when transiently expressed in tobacco via a virus vector . The symptom attenuation in the transgenic tobacco by the asymptomatic BC1 may involve trans-dominant negative interference.

J Bacteriol, 1997 Nov, 179(22), 7089 - 97
Hierarchical autoinduction in Ralstonia solanacearum: control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester; Flavier AB et al.; Bacteria employ autoinduction systems to sense the onset of appropriate cell density for expression of developmental genes . In many gram-negative bacteria, autoinduction involves the production of and response to diffusible acylated-homoserine lactones (acyl-HSLs) and is mediated by members of the LuxR and LuxI families . Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium that appears to autoregulate its virulence genes, produces compounds that promote expression of several heterologous acyl-HSL-responsive reporter gene constructs . High-pressure liquid chromatography of highly concentrated ethyl acetate extracts revealed that culture supernatants of strain AW1 contained two compounds with retention times similar to N-hexanoyl- and N-octanoyl-HSL . To investigate the role of these acyl-HSLs in R . solanacearum virulence gene expression, transposon mutants that were deficient for inducing an acyl-HSL-responsive reporter in Agrobacterium tumefaciens were generated . Three loci involved in normal acyl-HSL production were identified, one of which was shown to contain the divergently transcribed solR and solI genes, the luxR and luxI homologs, respectively . A 4.1-kb fragment containing solR and solI enabled all of the mutants (regardless of the locus inactivated) and a naturally acyl-HSL-defective strain of R . solanacearum to produce acyl-HSLs . Inactivation of solI abolished production of all detectable acyl-HSLs but affected neither the expression of virulence genes in culture nor the ability to wilt tomato plants . AW1 has a functional autoinduction system, because (i) expression of solI required SolR and acyl-HSL and (ii) expression of a gene linked to solR and solI, designated aidA, was acyl-HSL dependent . Because AidA has no homologs in the protein databases, its discovery provided no clues as to the role of acyl-HSLs in R . solanacearum gene regulation . However, expression of solR and solI required the global LysR-type virulence regulator PhcA, and both solR and solI exhibited a cell density-associated pattern of expression similar to other PhcA-regulated genes . The acyl-HSL-dependent autoinduction system in R . solanacearum is part of a more complex autoregulatory hierarchy, since the transcriptional activity of PhcA is itself controlled by a novel autoregulatory system that responds to 3-hydroxypalmitic acid methyl ester.

Mol Plant Microbe Interact, 1997 Nov, 10(8), 970 - 7
A transgenic mutant of Lactuca sativa (lettuce) with a T-DNA tightly linked to loss of downy mildew resistance; Okubara PA et al.; One hundred and ninety-two independent primary transformants of lettuce cv . Diana were obtained by co-cultivation with Agrobacterium tumefaciens carrying constructs containing maize Ac transposase and Ds . R2 families were screened for mutations at four genes (Dm) for resistance to downy mildew . One family, designated dm3t524, had lost resistance to an isolate of Bremia lactucae expressing the avirulence gene Avr3 . Loss of resistance segregated as a single recessive allele of Dm3 . The mutation was not due to a large deletion as all molecular markers flanking Dm3 were present . Loss of Dm3 activity co-segregated with a T-DNA from which Ds had excised . Genomic DNA flanking the right border of this T-DNA was isolated by inverse polymerase chain reaction . This genomic sequence was present in four to five copies in wild-type cv . Diana . One copy was missing in all eight deletion mutants of Dm3 and altered in dm3t524, indicating tight physical linkage to Dm3 . Three open reading frames (ORFs) occurred in a 6.6-kb region flanking the insertion site; however, expression of these ORFs was not detected . No similarities were detected between these ORFs and resistance genes cloned from other species . Transgenic complementation with 11-to 27-kb genomic fragments of Diana spanning the insertion site failed to restore Dm3 function to two ethyl methanesulfonate (EMS)-induced mutants of Dm3 or to cv . Cobham Green, which naturally lacks Dm3 activity . Therefore, either the T-DNA inserted extremely close to, but not within, Dm3 and the mutation may have been caused by secondary movement of Ds, or Dm3 activity is encoded by a gene extending beyond the fragments used for complementation.

J Bacteriol, 1997 Nov, 179(21), 6788 - 97
Interactions between heterologous FtsA and FtsZ proteins at the FtsZ ring; Ma X et al.; FtsZ and FtsA are essential for cell division in Escherichia coli and colocalize to the septal ring . One approach to determine what regions of FtsA and FtsZ are important for their interaction is to identify in vivo interactions between FtsA and FtsZ from different species . As a first step, the ftsA genes of Rhizobium meliloti and Agrobacterium tumefaciens were isolated and characterized . In addition, an FtsZ homolog that shared the unusual C-terminal extension of R . meliloti FtsZ1 was found in A . tumefaciens . In order to visualize their localization in cells, we tagged these proteins with green fluorescent protein (GFP) . When R . meliloti FtsZ1-GFP or A . tumefaciens FtsZ-GFP was expressed at low levels in E . coli, they specifically localized only to the E . coli FtsZ ring, possibly by coassembly . When A . tumefaciens FtsA-GFP or R . meliloti FtsA-GFP was expressed in E . coli, they failed to localize detectably to the E . coli FtsZ ring . However, when R . meliloti FtsZ1 was coexpressed with them, fluorescence localized to a band at the midcell division site, strongly suggesting that FtsA from either A . tumefaciens or R . meliloti can bind directly to its cognate FtsZ . As expected, GFP-tagged FtsZ1 and FtsA from either R . meliloti or A . tumefaciens localized to the division site in A . tumefaciens cells . Therefore, the 61 amino acid changes between A . tumefaciens FtsA and R . meliloti FtsA do not prevent their direct interaction with FtsZ1 from either species, suggesting that those residues are not essential for protein-protein contacts . Moreover, the failure of the two non-E . coli FtsA derivatives to interact strongly with E . coli FtsZ in this in vivo system unless their cognate FtsZ was also present suggests that FtsA-FtsZ interactions have coevolved and that the residues which differ between the E . coli proteins and those of the two other species may be important for specific interactions.

Appl Environ Microbiol, 1997 Oct, 63(10), 3825 - 30
Novel detoxification of the trichothecene mycotoxin deoxynivalenol by a soil bacterium isolated by enrichment culture; Shima J et al.; A mixed microbial culture capable of metabolizing deoxynivalenol was obtained from soil samples by an enrichment culture procedure . A bacterium (strain E3-39) isolated from the enrichment culture completely removed exogenously supplied deoxynivalenol from culture medium after incubation for 1 day . On the basis of morphological, physiological, and phylogenetic studies, strain E3-39 was classified as a bacterium belonging to the Agrobacterium-Rhizobium group . Thin-layer chromatographic analysis indicated the presence of one major and two minor metabolites of deoxynivalenol in ethyl acetate extracts of the E3-39 culture filtrates . The main metabolite was identified as 3-keto-4-deoxynivalenol by mass spectroscopy and 1H and 13C nuclear magnetic resonance analysis . The immunosuppressive toxicity of 3-keto-4-deoxynivalenol was evaluated by means of a bioassay based on the mitogen-induced and mitogen-free proliferations of mouse spleen lymphocytes . This compound exhibited a remarkably decreased (to less than one tenth) immunosuppressive toxicity relative to deoxynivalenol, indicating that the 3-OH group in deoxynivalenol is likely to be involved in exerting its immunosuppressive toxicity . Strain E3-39 was also capable of transforming 3-acetyldeoxynivalenol but not nivalenol and fusarenon-X.

Biosci Biotechnol Biochem, 1997 Sep, 61(9), 1459 - 64
Molecular cloning and nucleotide sequence of the arginase gene of Bacillus brevis TT02-8 and its expression in Escherichia coli; Shimotohno KW et al.; The gene from Bacillus brevis TT02-8 encoding arginase was cloned into Escherichia coli, and its nucleotide sequence was identified . The nucleotide sequence contained an open reading frame that encoded a polypeptide of 298 amino acid residues with a predicted molecular weight of 31,891, which was consistent with that previously calculated for arginase purified from this bacterium . Comparison of the deduced amino acid sequence of the B . brevis TT02-8 arginase with that of the prokaryotic and eukaryotic arginases of Bacillus caldovelox, Bacillus subtilis, Agrobacterium Ti plasmid C58, Saccharomyces cerevisiae, Coccidioides immitis, Xenopus laevis, Rana catesbeiana, rat liver, and human liver, showed 33-66% of the sequences to be similar; there were several highly conserved regions . Arginase activity was detected in Escherichia coli cells transformed with an expression plasmid of the cloned arginase gene.

Int J Syst Bacteriol, 1997 Oct, 47(4), 996 - 1006
Rhizobium gallicum sp . nov . and Rhizobium giardinii sp . nov., from Phaseolus vulgaris nodules; Amarger N et al.; Thirty-one strains of two new genomic species (genomic species 1 and 2) of rhizobia isolated from root nodules of Phaseolus vulgaris and originating from various locations in France were compared with reference strains of rhizobia by performing a numerical analysis of 64 phenotypic features . Each genomic species formed a distinct phenon and was separated from the other rhizobial species . A comparison of the complete 16S rRNA gene sequences of a representative of genomic species 1 (strain R602spT) and a representative of genomic species 2 (strain H152T) with the sequences of other rhizobia and related bacteria revealed that each genomic species formed a lineage independent of the lineages formed by the previously recognized species of rhizobia . Genomic species 1 clustered with the species that include the bean-nodulating rhizobia, Rhizobium leguminosarum, Rhizobium etli, and Rhizobium tropici, and branched with unclassified rhizobial strain OK50, which was isolated from root nodules of Pterocarpus klemmei in Japan . Genomic species 2 was distantly related to all other Rhizobium species and related taxa, and the most closely related organisms were Rhizobium galegae and several Agrobacterium species . On the basis of the results of phenotypic and phylogenetic analyses and genotypic data previously published and reviewed in this paper, two new species of the genus Rhizobium, Rhizobium gallicum and Rhizobium giardinii, are proposed for genomic species 1 and 2, respectively . Each species could be divided in two subgroups on the basis of symbiotic characteristics, as shown by phenotypic (host range and nitrogen fixation effectiveness) and genotypic data . For each species, one subgroup had the same symbiotic characteristics as R . leguminosarum biovar phaseoli and R . etli biovar phaseoli . The other subgroup had a species-specific symbiotic phenotype and genotype . Therefore, we propose that each species should be subdivided into two biovars, as follows: R . gallicum biovar gallicum and R . gallicum biovar phaseoli; and R . giardinii biovar giardinii and R . giardinii biovar phaseoli.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11726 - 30
T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes; Hansen G et al.; We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene . The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without virE2, together with a target plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences of 25 bp . We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome . The frequency of VirD1-VirD2-mediated integration events was about 20-35% of the total number of transformants . The addition of virE2 doubled the transformation efficiency . The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10723 - 8
Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein; Ballas N et al.; T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium . Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process . While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5' end . To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis . The identified protein, designated AtKAPalpha, specifically bound VirD2 in vivo and in vitro . VirD2-AtKAPalpha interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2 . The deduced amino acid sequence of AtKAPalpha was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin alpha family . Indeed, AtKAPalpha efficiently rescued a yeast mutant defective for nuclear import . Furthermore, AtKAPalpha specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.

Biochim Biophys Acta, 1997 Aug 29, 1336(2), 218 - 24
Purification and characterization of an extracellular lectin (Lectin I) from Agrobacterium radiobacter NCIM 2443; Joshi B et al.; A lectin from culture filtrate of Agrobacterium radiobacter NCIM 2443 is purified to homogeneity by ion exchange chromatography on a DEAE cellulose column followed by hydrophobic chromatography on phenyl sepharose and hydroxyapatite column chromatography . The protein (Lectin I) is a monomer of relative molecular mass 37,000, as determined by denaturing gel electrophoresis as well as size exclusion chromatography . Lectin I is stable at pH 5.0 and its isoelectric point is pH 4.0 . Amino acid analysis reveals that acidic amino acids and glycine are predominant amino acids and cysteine is absent in the lectin . Chemical modification of tryptophan residues causes more than 80% loss of haemagglutination activity of the lectin and 60% loss of activity is caused by modification of carboxyl groups . Lectin I agglutinates rabbit erythrocytes but does not agglutinate human A, B and O types of erythrocytes . It is specific for N-acetyl D-glucosamine, chitobiose, pNP-beta-mannoside as well as high mannose type glycopeptides . The relative inhibition by disaccharides, oligosaccharides and glycoproteins indicates that Lectin I recognizes Man3-GlcNAc-GlcNAc core carbohydrate structure of asparagine linked glycopeptides . Tobacco tissue extracts also inhibit the haemagglutination activity of Lectin I.

Mol Plant Microbe Interact, 1997 Sep, 10(7), 891 - 902
Characterization of two plasmid-borne lps beta loci of Rhizobium etli required for lipopolysaccharide synthesis and for optimal interaction with plants; Garcia-de los Santos A et al.; In Rhizobium etli CFN42, both the symbiotic plasmid (pd) and plasmid b (pb) are required for effective bean nodulation . This is due to the presence on pb of a region (lps beta) involved in lipopolysaccharide (LPS) biosynthesis . We report here the genetic array and functional features of this plasmid-borne region . The sequence analysis of a 3,595-bp fragment revealed the presence of a transcriptional unit integrated by two open reading frames (lps beta 1 and lps beta 2) essential for LPS biosynthesis and symblosis . The lps beta 1 encodes a putative 193 amino acid polypeptide that shows strong homology with glucosyl-1P and galactosyl-1P transferases . The deduced amino acid sequence of the protein encoded by lps beta 2 was very similar to that of proteins involved in surface polysaccharide biosynthesis, such as Pseudomonas aeruginosa WpbM, Bordetella pertussis BpIL, and Yersinia enterocolitica TrsG . DNA sequences homologous to lps beta 1 and lps beta 2 of R . etli CFN42 were consistently found in functionally equivalent plasmids of R . etli, R . leguminosarum bv . viciae, and R . leguminosarum hv . trifolii strains, but not in R . meliloti, R . loti, R . tropici, R . fredii, Bradyrhizobium, Azorhizobium, and Agrobacterium tumefaciens . Even though Rhizobium and Agrobacterium do not share lps beta sequences, their presence is required for crown-gall tumor induction by R . etli transconjugants carrying the Ti plasmid.

Mol Plant Microbe Interact, 1997 Sep, 10(7), 852 - 60
Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris; Desiderio A et al.; The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P . Toubart, A . Desiderio, G . Salvi, F . Cervone, L . Daroda, G . De Lorenzo, C . Bergmann, A . G . Darvill, and P . Albersheim, Plant J . 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation . Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f . sp . lycopersici, Botrytis cinerea, and Alternaria solani . No evident enhanced resistance, compared with the resistance of untransformed plants, was observed . The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector . PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N . benthamiana plants were tested with several fungal polygalacturonases (PGs) . PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P . vulgaris (bulk bean PGIP) . Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme . Moreover, PGIP-1 expressed in tomato and N . benthamiana had only a limited capacity to inhibit crude PG preparations from F . oxysporum f . sp . lycopersici, B . cinerea, and A . solani . Differential affinity chromatography was used to separate PGIP proteins present in P . vulgaris extracts . A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F . moniliforme PGs . Our data show that PGIPs with different specificities are expressed in P . vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.

J Mol Biol, 1997 Sep 5, 271(5), 718 - 27
The molecular structure of agrobacterium VirE2-single stranded DNA complexes involved in nuclear import; Citovsky V et al.; Nuclear import of DNA is a central event in genetic transformation of plant cells by Agrobacterium tumefaciens . Agrobacterium elicits tumors on plant hosts by transporting a single-stranded (ss) copy of the bacterial transferred DNA (T-DNA) from its Ti (tumor-inducing) plasmid into the plant cell nucleus . Presumably, the process of T-DNA nuclear import is mediated by two agrobacterium proteins, VirD2 and VirE2, which are thought to directly associate with the transported T-DNA . Both proteins have been shown to contain functional nuclear localizations signals (NLS) . Recently, VirE2 alone has been shown to actively transport ssDNA into the plant cell nucleus . To understand the process of DNA nuclear import, it is important to know the structure of the transport intermediate . To this end, complexes of VirE2 and ssDNA were analyzed by scanning transmission electron microscopy (STEM) . This analysis suggests that VirE2 packages ssDNA into semi-rigid, hollow cylindrical filaments with a telephone cord-like coiled structure . The outer diameter of these complexes is too large to enter the nucleus by diffusion but is within the size exclusion limits of the active nuclear import . Detailed mass analysis of VirE2-ssDNA filaments is presented and a structural model is proposed .

J Bacteriol, 1997 Sep, 179(18), 5869 - 77
The CcrM DNA methyltransferase is widespread in the alpha subdivision of proteobacteria, and its essential functions are conserved in Rhizobium meliloti and Caulobacter crescentus; Wright R et al.; The Caulobacter crescentus DNA methyltransferase CcrM (M.CcrMI) methylates the adenine residue in the sequence GANTC . The CcrM DNA methyltransferase is essential for viability, but it does not appear to be part of a DNA restriction-modification system . CcrM homologs are widespread in the alpha subdivision of gram-negative bacteria . We have amplified and sequenced a 258-bp region of the cerM gene from several of these bacteria, including Rhizobium meliloti, Brucella abortus, Agrobacterium tumefaciens, and Rhodobacter capsulatus . Alignment of the deduced amino acid sequences revealed that these proteins constitute a highly conserved DNA methyltransferase family . Isolation of the full-length ccrM genes from the aquatic bacterium C . crescentus, the soil bacterium R . meliloti, and the intracellular pathogen B . abortus showed that this sequence conservation extends over the entire protein . In at least two alpha subdivision bacteria, R . meliloti and C . crescentus, CcrM-mediated methylation has important cellular functions . In both organisms, CcrM is essential for viability . Overexpression of CcrM in either bacterium results in defects in cell division and cell morphology and in the initiation of DNA replication . Finally, the C . crescentus and R . meliloti ccrM genes are functionally interchangeable, as the complemented strains are viable and the chromosomes are methylated . Thus, in both R . meliloti and C . crescentus, CcrM methylation is an integral component of the cell cycle . We speculate that CcrM-mediated DNA methylation is likely to have similar roles among alpha subdivision bacteria.

J Bacteriol, 1997 Sep, 179(18), 5835 - 42
Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins; Zhou XR et al.; The Agrobacterium tumefaciens VirB11 ATPase is postulated to assemble with VirB proteins and the VirD4 protein into a transport system which is dedicated to the export of oncogenic nucleoprotein particles to plant cells . To gain genetic evidence for interactions between VirB11 and other subunits of this transport system, we screened a PCR-mutagenized virB11 library for alleles that diminish the virulence of the wild-type strain A348 . Two classes of alleles displaying negative dominance were identified . One class failed to complement a delta virB11 mutation, indicating that the corresponding mutant proteins are nonfunctional . The second class complemented the delta virB11 mutation, indicating that the mutant proteins are fully functional in strains devoid of native VirB11 . Mutations of both classes of alleles were in codons for residues clustered in two regions of VirB11, both located outside the Walker A nucleotide binding motif . All dominant alleles were suppressed at least to some extent by multicopy expression of the virB9, virB10, and/or virB11 genes . Taken together, results of these investigations indicate that (i) a functional T-complex transporter is composed of more than one VirB11 subunit and (ii) VirB11 undergoes complex formation with VirB9 and VirB10 during transporter biogenesis.

Plant Mol Biol, 1997 Sep, 35(1-2), 205 - 18
Transformation of rice mediated by Agrobacterium tumefaciens; Hiei Y et al.; Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall . Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently . Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996 . A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes . It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing 'competent' cells are infected . The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance . Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants . Delivery of foreign DNA to rice plants via A . tumefaciens is a routine technique in a growing number of laboratories . This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.

Plant Mol Biol, 1997 Sep, 35(1-2), 197 - 203
Rice transformation: bombardment; Christou P; Bombardment-based methodology is responsible for the effective genetic manipulation of major cereals including rice . Many groups reported significant advances on various aspects of rice molecular biology and genetic engineering using procedures based on bombardment technology . Molecular and genetic characterization of large numbers of these plants (more than 500 independent transgenic plants) provided information on structure, expression and stability of integrated DNA through multiple generations . Such evaluations were carried out in the greenhouse and in the field . Stability of expression was found to be dependent on the nature of the promoter and the transgene, and in specific cases on gene copy number . Direct DNA transfer utilizing particle bombardment for the delivery of foreign DNA into rice tissue results in the recovery of large numbers of independently derived transgenic plants in a variety-independent fashion . Gene copy number, level and stability of expression of transgenes can be compared to other DNA delivery methods, direct or indirect, including Agrobacterium-mediated gene transfer . In this paper, the technology is summarized and discussed in terms of present and future applications, including field trials and potential commercialization of transgenic rice expressing a number of genes of agronomic interest such as pest and herbicide resistance.

Plant Mol Biol, 1997 Aug, 34(6), 913 - 22
Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation; Wenck A et al.; When Agrobacterium was used to transform Nicotiana plumbaginifolia protoplasts and Arabidopsis thaliana roots and seedlings, a large number of plants were found in which not only the T-region defined by the border repeat sequences but the entire binary vector was integrated, as determined by both PCR and Southern analysis techniques . N . plumbaginifolia protoplast co-cultivation experiments yielded 3 out of 5 transformants with collinear sequence past the left border . In Arabidopsis root transformation experiments, 33% (6/18) of the transformants had T-DNA which exceeded the left border repeat . Vacuum infiltration of Arabidopsis seedlings produced even a greater percentage of transformants with sequences outside the left border repeat (62%, 39/63) . The long transfer DNA cosegregated with the T-region encoded hygromycin resistance in the T2 progeny eliminating the possibility that long transfer DNA was of extrachromosomal or Agrobacterium origin . The high frequency of long transfer after vacuum infiltration of A . thaliana needs to be considered when analyzing T-DNA tagged mutants.

J Formos Med Assoc, 1997 Aug, 96(8), 664 - 6
Agrobacterium radiobacter bacteremia in a patient with chronic obstructive pulmonary disease; Yu WL et al.; Agrobacterium radiobacter is a gram-negative bacillus, which is recognized as an emerging opportunistic human pathogen . To our knowledge, there have been only 25 cases of A . radiobacter bacteremia reported . In most of these, A . radiobacter was associated with long-term indwelling plastic central venous catheters . We describe a 78-year-old man who had a history of chronic obstructive pulmonary disease with long-term use of a corticosteroid . He was admitted to the China Medical College Hospital with pneumonia caused by Serratia marcescens . His general condition gradually improved after initiation of appropriate treatment . Unfortunately, he developed A . radiobacter bacteremia while hospitalized in the medical intensive care unit . With the onset of this infection, the patient had a high fever, leukocytosis, raised C-reactive protein level, and positive blood cultures for A . radiobacter . A central venous catheter-related infection was suspected because of redness and localized tenderness at the catheter site . The patient gradually recovered after removal of the catheter and appropriate antimicrobial treatment with latamoxef 1.5 g intravenously every 8 hours for 10 days.

J Bacteriol, 1997 Sep, 179(17), 5372 - 9
Attachment of Agrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide; Reuhs BL et al.; An early step in crown gall tumor formation involves the attachment of Agrobacterium tumefaciens to host plant cells . A . tumefaciens C58::A205 (C58 attR) is a Tn3HoHo1 insertion mutant that was found to be avirulent on Bryophyllum daigremontiana and unable to attach to carrot suspension cells . The mutation mapped to an open reading frame encoding a putative protein of 247 amino acids which has significant homology to transacetylases from many bacteria . Biochemical analysis of polysaccharide extracts from wild-type strain C58 and the C58::A205 mutant showed that the latter was deficient in the production of a cell-associated polysaccharide . Anion-exchange chromatography followed by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry analyses showed that the polysaccharide produced by strain C58 was an acetylated, acidic polysaccharide and that the polysaccharide preparation contained three sugars: glucose, glucosamine, and an unidentified deoxy-sugar . Application of the polysaccharide preparation from strain C58 to carrot suspension cells prior to inoculation with the bacteria effectively inhibited attachment of the bacteria to the carrot cells, whereas an identical preparation from strain C58::A205 had no inhibitory effect and did not contain the acidic polysaccharide . Similarly, preincubation of Arabidopsis thaliana root segments with the polysaccharide prevented attachment of strain C58 to that plant . This indicates that the acidic polysaccharide may play a role in the attachment of A . tumefaciens to host soma plant cells.

Curr Microbiol, 1997 Sep, 35(3), 145 - 50
Transposon-induced catalase-deficient Agrobacterium tumefaciens; Khetmalas MB; Agrobacterium tumefaciens MKR, a nonpathogenic strain, has three catalase isozymes and one superoxide dismutase but no detectable peroxidase activity . A large number (8400) of transconjugants were obtained with pSUP1011::Tn5 suicide vector . The transposition frequencies were found to be greater in biparental mating than in triparental mating with helper plasmid . Mutants MLA31, MLA32, MLA41, and MLA41(a), generated by transposon mutagenesis, all lacked one of the catalase isozymes . Mutants were more susceptible to cell death than the wild type upon direct exposure to 10.0 mmol L-1 H2O2 . The specific activity of the enzyme catalase was found to be higher in nitrogen-rich growth medium than carbon-rich growth medium.

Microbiology, 1997 Aug, 143 ( Pt 8), 2825 - 31
Sequence, localization and characteristics of the replicator region of the symbiotic plasmid of Rhizobium etli; Ramirez-Romero MA et al.; The replicator region of the symbiotic plasmid of Rhizobium etli CFN42 was cloned and sequenced . A plasmid derivative (pH3) harbouring a 5-6 kb HindIII fragment from the symbiotic plasmid was found to be capable of independent replication and eliminated the symbiotic plasmid when introduced into a R . etli CFNX101 strain (a recA derivative) . The stability and the copy number of pH3 were the same as that of the symbiotic plasmid, indicating that the information required for stable replication and incompatibility resides in the 5.6 kb HindIII fragment . The sequence analysis of this fragment showed the presence of three ORFs similar in sequence analysis of this fragment showed the presence of three ORFs similar in sequence and organization to repA, repB and repC described for the replicator regions of the Agrobacterium plasmids pTiB653 and pRiA4b and for the R . leguminosarum cryptic plasmid pRL8JI . Hybridization studies showed that p42d-like replicator sequences are found in the symbiotic plasmids of other R . etli strains and in a 'cryptic' plasmid of R . tropici.

J Bacteriol, 1997 Aug, 179(15), 4946 - 8
Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1; Chistoserdova L et al.; A gene (gckA) responsible for the activity of glycerate kinase has been identified within a chromosomal fragment of the serine cycle methylotroph Methylobacterium extorquens AM1 . A mutation in gckA leads to a specific C1-negative phenotype . The polypeptide sequence derived from gckA showed high similarity to a product of ttuD essential for tartrate metabolism in Agrobacterium vitis . Our data suggest that gckA and ttuD might be structural genes for glycerate kinase and that the serine cycle and the tartrate utilization pathway share a series of reactions.

J Bacteriol, 1997 Aug, 179(15), 4831 - 40
A T-DNA gene required for agropine biosynthesis by transformed plants is functionally and evolutionarily related to a Ti plasmid gene required for catabolism of agropine by Agrobacterium strains; Hong SB et al.; The mechanisms that ensure that Ti plasmid T-DNA genes encoding proteins involved in the biosynthesis of opines in crown gall tumors are always matched by Ti plasmid genes conferring the ability to catabolize that set of opines on the inducing Agrobacterium strains are unknown . The pathway for the biosynthesis of the opine agropine is thought to require an enzyme, mannopine cyclase, coded for by the ags gene located in the T(R) region of octopine-type Ti plasmids . Extracts prepared from agropine-type tumors contained an activity that cyclized mannopine to agropine . Tumor cells containing a T region in which ags was mutated lacked this activity and did not contain agropine . Expression of ags from the lac promoter conferred mannopine-lactonizing activity on Escherichia coli . Agrobacterium tumefaciens strains harboring an octopine-type Ti plasmid exhibit a similar activity which is not coded for by ags . Analysis of the DNA sequence of the gene encoding this activity, called agcA, showed it to be about 60% identical to T-DNA ags genes . Relatedness decreased abruptly in the 5' and 3' untranslated regions of the genes . ags is preceded by a promoter that functions only in the plant . Expression analysis showed that agcA also is preceded by its own promoter, which is active in the bacterium . Translation of agcA yielded a protein of about 45 kDa, consistent with the size predicted from the DNA sequence . Antibodies raised against the agcA product cross-reacted with the anabolic enzyme . These results indicate that the agropine system arose by a duplication of a progenitor gene, one copy of which became associated with the T-DNA and the other copy of which remained associated with the bacterium.

Transgenic Res, 1997 Jul, 6(4), 289 - 96
Expression of the human milk protein beta-casein in transgenic potato plants; Chong DK et al.; A 1177 bp cDNA fragment encoding the human milk protein beta-casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase (mas1',2') promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods . Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human beta-casein cDNA . The presence of human beta-casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments . Human beta-casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis . Human beta-casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis . Human beta-casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa . Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as beta-casein . The above experiments demonstrate the expression of human milk beta-casein as part of an edible food plant . These findings open the way for reconstitution of human milk in edible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children.

South Med J, 1997 Jul, 90(7), 752 - 4
Cellulitis and myositis caused by Agrobacterium radiobacter and Haemophilus parainfluenzae after influenza virus vaccination; Owensby JE et al.; Agrobacterium radiobacter is a gram-negative aerobic bacillus that has been reported as a cause of disease only 36 times in the literature . More than half of the patients (25) have had bacteremia . Peritonitis, urinary tract infection, endocarditis, and one case of cellulitis associated with bacteremia have also been reported . Infection is often associated with immunosuppression and the presence of a plastic foreign body, such as central venous catheters, nephrostomy tubes, intraperitoneal catheters, and prosthetic cardiac valves . We present apparently the first case of A radiobacter causing myositis after influenza virus vaccination.

Mol Plant Microbe Interact, 1997 Jul, 10(5), 550 - 9
Identification and characterization of a gene on Rhizobium meliloti pSyma, syrB, that negatively affects syrM expression; Barnett MJ et al.; The Rhizobium meliloti SyrM protein activates transcription of nodD3 and syrA . Regulation of syrM is complex and may involve as yet undiscovered genes . Here we report the isolation of insertion mutants showing increased expression of a syrM-gusA gene fusion . Characterization of one mutant strain, designated SYR-B, revealed a mutation consisting of a transposon insertion linked to a large deletion . The corresponding wild-type DNA was cloned as a 5.3-kb BamHI fragment . Genetic and physical analysis of this DNA demonstrated that an open reading frame (ORF) near one end of the fragment, encoding the 16.5-kDa SyrB protein, is responsible for the repression of syrM activity . Results of complementation experiments with the 5.3-kb BamHI DNA led us to hypothesize that other genes within this DNA fragment interfere with the expression or activity of SyrB . Our analysis showed that the region upstream of syrB contains three ORFs . One ORF is similar to the Ros repressor of Agrobacterium tumefaciens and the MucR repressor of R . meliloti.

Proc Natl Acad Sci U S A, 1997 Jun 10, 94(12), 6036 - 41
Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography; Shaw PD et al.; Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment . We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction . With the exception of N-butanoyl-L-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested . The intensity of the response was proportional to the amount of the signal molecule chromatographed . Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility . Using the assay, we showed that some bacteria produce as many as five detectable signal molecules . Structures could be assigned tentatively on the basis of mobility and spot shape . The dominant species produced by Pseudomonas syringae pv . tabaci chromatographed with the properties of N-(3-oxohexanoyl)-L-homoserine lactone, a structure that was confirmed by mass spectrometry . An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties . These were identified as the 3-hydroxy- forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-L-homoserine lactone . The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules.

J Biol Chem, 1997 Jun 6, 272(23), 14650 - 7
Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1; Rink R et al.; The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme . The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa . An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A . radiobacter CFZ11 . The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin . Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the alpha/beta-hydrolase fold family . This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy . The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246 . Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin . The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His275 --> Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.

Can J Microbiol, 1997 Jun, 43(6), 526 - 33
Comparison of partial 23S rDNA sequences from Rhizobium species; Tesfaye M et al.; A hypervariable region of Rhizobium 23S rDNA was amplified by polymerase chain reaction and phylogenetic relationships of several strains were determined by comparing nucleotide sequences of the amplified product . Variation in the 23S rDNA nucleotide sequences was consistent with phylogenetic relationships determined by host nodulation specificity and (or) 16S rDNA sequence analysis . Six strains representing three Rhizobium species (R . leguminosarum bv . trifolii, R . meliloti, and R . etli), and two strains each of Bradyrhizobium and Agrobacterium were clustered into five rDNA groups . Unique features identified by secondary structure analysis of the 23S rRNA sequenced region were consistent with the hypothesis that 23S rDNA could be used to design species- or strain-specific Rhizobium probes.

Plant J, 1997 Jun, 11(6), 1315 - 24
Phosphorylation of nuclear proteins directs binding to salicylic acid-responsive elements; Stange C et al.; The cis-located DNA sequence as-1 (Activation Sequence-1) from CaMV 35S promoter has been previously identified as an element that can confer inducibility by salicylic acid (SA) with immediate early kinetics . This sequence specifically binds to ASF-1 (Activation Sequence Factor-1), previously characterized in tobacco nuclear extracts . To assess whether modulation of ASF-1 binding activity can explain the activation of the as-1 sequence observed in vivo, we performed electrophoretic mobility shift assays using nuclear extracts from SA-treated and water-treated tobacco plants . Our results indicate that treatment of plants with SA increases ASF-1 binding to as-1 and to ocs, an as-1-like element from the Agrobacterium octopine synthase gene . In contrast, SA treatment has no effect on the binding of GT-1 factor to its target light-inducible box II element . Furthermore, treatment of nuclear extracts from SA-treated plants with alkaline phosphatase decreases ASF-1 binding to the as-1 element . This can be reversed by pretreatment with 10 mM NaF . Accordingly, pretreatment of nuclear extracts from control water-treated plants with ATP produces an increase in ASF-1 binding activity similar to that observed with SA . This effect of ATP is reversed by treatment with alkaline phosphatase and prevented by quercetin, a casein kinase II inhibitor . These results support the hypothesis that a nuclear protein kinase is involved in the immediate early events of transcriptional activation triggered by SA.

Biosci Biotechnol Biochem, 1997 Jun, 61(6), 1055 - 8
Purification and properties of a new enzyme, D-carnitine dehydrogenase, from Agrobacterium sp . 525a; Setyahadi S et al.; A new enzyme, D-carnitine dehydrogenase from Agrobacterium sp . 525a, was purified by DEAE-Toyopearl, ammonium sulfate fractionation, Sephadex G-75, affinity chromatography, and Mono Q and TSK-gel filtration column chromatography . The enzyme had the molecular mass of 89 kDa and consisted of three identical subunits . The optimum pH for the oxidation reaction was 9.3 . The Michaelis constants for D-carnitine and NAD+ were 3.1 and 0.07 mM, respectively . The N-terminal 20 amino acids were sequenced.

J Bacteriol, 1997 Jun, 179(11), 3404 - 9
Delineation of the interaction domains of Agrobacterium tumefaciens VirB7 and VirB9 by use of the yeast two-hybrid assay; Das A et al.; The Agrobacterium tumefaciens VirB proteins are postulated to form a transport pore for the transfer of T-DNA . Formation of the transport pore will involve interactions among the VirB proteins . A powerful genetic method to study protein-protein interaction is the yeast two-hybrid assay . To test whether this method can be used to study interactions among the VirB membrane proteins, we studied the interaction of VirB7 and VirB9 in yeast . We recently demonstrated that VirB7 and VirB9 form a protein complex linked by a disulfide bond between cysteine 24 of VirB7 and cysteine 262 of VirB9 (L . Anderson, A . Hertzel, and A . Das, Proc . Natl . Acad . Sci . USA 93:8889-8894, 1996) . We now demonstrate that VirB7 and VirB9 interact in yeast, and this interaction does not require the cysteine residues essential for the disulfide linkage . By using defined segments in fusion constructions, we mapped the VirB7 interaction domain of VirB9 to residues 173 to 275 . In tumor formation assays, both virB7C24S and virB9C262S expressed from a multicopy plasmid complemented the respective deletion mutation, indicating that the cysteine residues may not be essential for DNA transfer.

Nature, 1997 May 22, 387(6631), 394 - 401
Molecular basis of symbiosis between Rhizobium and legumes; Freiberg C et al.; Access to mineral nitrogen often limits plant growth, and so symbiotic relationships have evolved between plants and a variety of nitrogen-fixing organisms . These associations are responsible for reducing 120 million tonnes of atmospheric nitrogen to ammonia each year . In agriculture, independence from nitrogenous fertilizers expands crop production and minimizes pollution of water tables, lakes and rivers . Here we present the complete nucleotide sequence and gene complement of the plasmid from Rhizobium sp . NGR234 that endows the bacterium with the ability to associate symbiotically with leguminous plants . In conjunction with transcriptional analyses, these data demonstrate the presence of new symbiotic loci and signalling mechanisms . The sequence and organization of genes involved in replication and conjugal transfer are similar to those of Agrobacterium, suggesting a recent lateral transfer of genetic information.

Mol Gen Genet, 1997 May 20, 254(5), 529 - 38
The regulatory protein MucR binds to a short DNA region located upstream of the mucR coding region in Rhizobium meliloti; Bertram-Drogatz PA et al.; The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan . The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system . Cell extracts of the production strain contained about 20% of a polypeptide of 17 kDa apparent molecular mass, corresponding to the size expected for MucR . As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene . Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene . Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site . Although binding of MucR to this site exhibited a moderate dissociation constant of Kd approximately 1.4 x 10(-7) M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding.

Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4861 - 5
Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes; Yang X et al.; A hammerhead ribozyme {R(-)} targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme {mR(-)} were designed, cloned, and transcribed . As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50 degrees C . The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2 . Transgenic potato plants (cultivar Desiree) were generated by Agrobacterium tumefaciens-mediated transformation . Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation . In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector . The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Appl Microbiol Biotechnol, 1997 May, 47(5), 560 - 5
3-Ketoglycoside-mediated metabolism of sucrose in E . coli as conferred by genes from Agrobacterium tumefaciens; Schuerman PL et al.; Escherichia coli strains that did not have the ability to use sucrose as a sole carbon source gained this ability after receiving a cloned fragment of DNA from Agrobacterium tumefaciens . No invertase was detected in the sucrose-metabolizing E . coli, but evidence for the activity of certain enzymes, known to be produced by biotype 1 strains of Agrobacterium, were found . Evidence was found for the presence of D-glucoside 3-dehydrogenase (G3DH) and alpha-3-ketoglucosidase . The activity of enzyme extracts on 3-ketosucrose also indicated that 3-ketoglucose reductase, or some enzyme that acts on 3-ketoglucose, was present in the Suc+ E . coli as well . The fragment was found to complement a G3DH mutant of A . tumefaciens and was also found to confer chemotaxis towards sucrose in E . coli.

Plant Mol Biol, 1997 May, 34(2), 357 - 62
Expression of human muscarinic cholinergic receptors in tobacco; Mu JH et al.; We expressed human m1, m2 and chimeric muscarinic cholinergic receptors (MAChR) in tobacco plants and in cultured BY2 tobacco cells using Agrobacterium-mediated transformation . The membranes of most transgenic plants and calli bound muscarinic ligands with appropriate affinities, kinetics and pharmacologic specificity, as determined by direct and competitive binding measurements using the muscarinic ligand {3H}quinuclidinyl benzylate (QNB) . Membranes of untransformed plants and calli or those transformed with vector alone did not bind {3H}QNB . Preliminary experiments did not suggest regulation of endogenous plant G protein signalling pathways by the recombinant receptors . Membranes from one callus clone expressed m1 MAChR at the level of 2.0-2.5 pmol {3H}QNB bound per mg membrane protein, more than the number of m1 MAChR in mammalian brain and comparable to that expressed in Sf9 insect cells using baculovirus vectors . This work demonstrates high level expression of active G protein-coupled receptors in plants, such that signaling might be genetically reconstituted by co-expression of appropriate G proteins and effectors.

Differentiation, 1997 May, 61(4), 213 - 21
Tobacco plants carrying a tms locus of Ti-plasmid origin and the Hl-1 allele are tumor prone; Meyer AD et al.; The autonomous growth of plant tumor cells is believed to result from their persistent loss of the requirement for growth hormones such as auxin and cytokinin . The partially dominant gene Habituated leaf-1 (Hl-1) regulates the requirement of cultures tissues of Havana 425 tobacco (Nicotiana tabacum L.) for cytokinins . The Hl-1 allele can partially restore the tumor phenotype in tobacco cells transformed with a Agrobacterium tumefaciens Ti plasmid defective in the isopentenyl transferase locus, which encodes a key enzyme in cytokinin biosynthesis and is required for neoplastic growth . To investigate the oncogenic function of Hl-1, we transformed wild-type (hl-1/hl-1) and Hl-1/Hl-1 tobacco plants with the tms locus derived from the limited-host-range Ti plasmid pTiAg162 . This locus encodes enzymes for biosynthesis of the auxin indole-3-acetic acid . Grafting tests and measurements of the hormone requirement of cultured explants show that wound-induced overgrowths arising in tms transformed Hl-1 plants are tumorous . While some wound-induced overgrowths also formed in hl-1/hl-1 transformants, these showed slight hormone-autotrophic growth and weak tumorigenicity in grafting tests . In addition, Hl-1/Hl-1 tms/tms plants, but not hl-1/hl-1 tms/tms plants, spontaneously developed rooty teratomatous overgrowths, showed flowering abnormalities, and formed calli at the base of the stem in young seedlings . Thus, Hl-1 tms plants exhibit a tumor-prone phenotype, and in this regard closely resemble tumor-prone hybrids that arise in certain interspecific crosses of Nicotiana species . Our results show that the interaction of just two genetic elements-the mutant Hl-1 allele of the tobacco host with tms genes of Ti plasmid origin-are sufficient for a tumor-prone phenotype.

J Infect, 1997 May, 34(3), 215 - 8
Broviac catheter-related bacteraemias due to unusual pathogens in children with cancer: case reports with literature review; Castagnola E et al.; Among 102 episodes of intravenous catheter related bacteraemias documented between January 1989 and July 1996 in children receiving antineoplastic chemotherapy or bone marrow transplantation at G . Gaslini Children's Hospital, Genoa, Italy, were identified seven episodes due to unusual pathogens: Bacillus circulans, Bacillus licheniformis, Brevibacterium casei, Flavimonas oryzihabitans, Porphyromonas asaccharolytica, Comamonas acidovorans and Agrobacterium radiobacter . Susceptibility to different antibiotics of all strains are reported . In all cases catheter removal was required for culture negativization . All episodes were diagnosed in absence of granulocytopenia.

Plant J, 1997 May, 11(5), 1007 - 16
Ribosomal transcription units integrated via T-DNA transformation associate with the nucleolus and do not require upstream repeat sequences for activity in Arabidopsis thaliana; Wanzenbock EM et al.; Ribosomal repeat units of Arabidopsis thaliana were introduced into the A . thaliana genome via Agrobacterium-mediated transformation . Ribosomal transgenes integrated into chromosomal regions outside the nucleolus organizers . Cytological data suggest that the transgenes associate with a nucleolus . To allow detection of transgenic rRNA, a short extension was inserted into the V1 variable region of the 25S ribosomal gene . The RNA transcript from the transgene undergoes a series of maturation steps, including correct processing of the 5' end of 25S rRNA . Using primer extension analysis, expression of a complete rDNA repeat unit was compared with the activity of a repeat unit lacking a sequence called 'upstream Sal repeats' . No qualitative or quantitative differences were detected, suggesting that upstream repeat sequences of the rDNA intergenic region do not act as transcriptional enhancers for RNA polymerase L in A . thaliana.

Plant J, 1997 May, 11(5), 945 - 57
Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: evidence for multiple complex patterns of integration; Kononov ME et al.; During the process of crown gall tumorigenesis, Agrobacterium tumefaciens transfers part of the tumor-inducing (Ti) plasmid, the T-DNA, to a plant cell where it eventually becomes stably integrated into the plant genome . Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA . The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector' system; this system is frequently used to generate transgenic plants . Scientists initially thought that only those sequences located between T-DNA left and right borders transferred to the plant . More recently, however, several reports have appeared describing the integration of the non-T-DNA binary vector 'backbone' sequences into the genome of transgenic plants . In order to investigate this phenomenon, we constructed T-DNA binary vectors containing a nos-nptll gene within the T-DNA and a mas2'-gusA (beta-glucuronidase) gene outside the T-DNA borders . We regenerated kanamycin-resistant transgenic tobacco plants and analyzed these plants for the expression of the vector-localized gusA gene and for the presence of binary vector backbone sequences . Approximately one-fifth of the plants expressed detectable GUS activity . PCR analysis indicated that approximately 75% of the plants contained the gusA gene . Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T-DNA border . The vector backbone sequences could also integrate into the plant genome independently of (unlinked to) the T-DNA . Although we could readily detect T-strands containing the T-DNA within the bacterium, we could not detect T-strands containing only the vector backbone sequences or these vector sequences linked to the T-DNA.

Microbiology, 1997 May, 143 ( Pt 5), 1513 - 20
Identification by PCR of genes encoding multiple response regulators; Morel-Deville F et al.; Environmental sensing in bacteria often involves the concerted action of sensor kinases and response regulators . Degenerate oligonucleotide primers were designed on the basis of amino acid similarity in the response regulators of these two-component systems . The primers were used in PCR to specifically amplify an internal DNA segment corresponding to the receiver module domain from genes encoding response regulators . Amplification products of the expected size were obtained from 12 different Gram-positive and Gram-negative bacteria . Sequence analysis revealed that 22 DNA fragments, which clearly originated from response regulator genes, were amplified from Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis and Lactobacillus bulgaricus . In each of these four species the receiver module of putative response regulator genes, which do not seem to be related to any of the already characterized genes, was identified . This simple and powerful method is therefore particularly useful for discovering new signal transduction systems which cannot be revealed by usual genetic studies.

Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4794 - 9
A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants; Brummell DA et al.; Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone . All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall . Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins . The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation . Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated . Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation . The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes . EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis . The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

Mol Gen Genet, 1997 Apr 16, 254(3), 337 - 43
Wound-inducible and organ-specific expression of ORF13 from Agrobacterium rhizogenes 8196 T-DNA in transgenic tobacco plants; Hansen G et al.; Tissue-specific expression of the ORF13 promoter from Agrobacterium rhizogenes 8196 was assessed throughout the development of transgenic tobacco plants using a GUS reporter gene . ORF13 exhibited high activity in roots but with different patterns of expression . The activity of the ORF13 promoter in vascular tissues increased from the base to the tip of the stem . The ORF13 promoter is wound inducible in a limited area adjacent to the wound site . The time course of wound induction of ORF13 in transgenic tobacco containing an ORF13 promoter-GUS translational fusion was similar to that previously described for genes involved in plant defense responses . A series of 5' deletions of the ORF13 promoter fused to the beta-glucuronidase gene was examined for expression in roots and leaves of transgenic plants . Cis-acting elements that modulate quantitative expression of the transgene after wounding were detected.

Gene, 1997 Apr 11, 189(1), 139 - 41
The Agrobacterium tumefaciens motor gene, motA, is in a linked cluster with the flagellar switch protein genes, fliG, fliM and fliN; Deakin WJ et al.; We report the sequence of 3978 bp of the Agrobacterium tumefaciens chromosome which contains a putative operon encoding the homologues of the transmembrane proton channel protein MotA, and the flagellar switch proteins FliM, FliN and FliG . Two transposon insertions in fliG result in a non-flagellate phenotype, indicating that this gene at least is required for flagellar assembly.

Gene, 1997 Apr 11, 189(1), 135 - 7
Isolation and characterisation of a linked cluster of genes from Agrobacterium tumefaciens encoding proteins involved in flagellar basal-body structure; Deakin WJ et al.; We report the DNA sequence of 7205 bp of the Agrobacterium tumefaciens chromosome . This contains a putative operon encoding homologues of the flagellar rod and associated proteins FlgBCG and FliE, the L and P ring proteins (FlgHI) a possible flagellum-specific export protein FliP, and two proteins of unknown function, FlgA and FliL . Several of these genes have overlapping stop and start codons . Three non-flagellate Tn5-induced mutations map to this operon: fla-11 to the first gene, encoding the rod protein FlgB; fla-15 to flgA; and fla-12 to fliL . A site-specific mutation introduced into the final gene in this cluster, fliP, also resulted in a non-flagellate phenotype . This indicates that the operon is expressed, and that at least FlgB, FlgA, FliL and FliP are required for flagellar assembly in A . tumefaciens . The bulk of this operon is conserved in the same order in Rhizobium meliloti.

Biochemistry (Mosc), 1997 Apr, 62(4), 433 - 9
Tryptophan synthase from Agrobacterium tumefaciens 8628: isolation and properties; Rekoslavskaya NI et al.; Tryptophan synthase was isolated from a highly virulent strain of Agrobacterium tumefaciens 8628 (octopine type) . Separation of tryptophan synthase from thermolabile protease was accomplished using fractionation with polyethylene glycol-6000 followed by ion-exchange chromatography with a pH gradient . Molecular weights of alpha- and beta-subunits are 33 and 51 kD, respectively . The tryptophan synthase is stable at 60 degrees C because of heat-tolerance beta-subunits . After heating the activity of tryptophan synthase increased up to 20 times while temperature-labile proteases lost their activities . Reaction with antibodies showed the presence of four protein bands, one of which was coeluted with nucleic acids during ion-exchange chromatography . It is suggested that the basic tryptophan synthase is encoded by trp genes in a plasmid and its role is to provide the precursor with the prokaryotic pathway of indole-3-acetic acid biosynthesis, which determines the virulence of A . tumefaciens . There is perhaps a cooperation between iaaM, iaaH, and trp genes in the plasmid during plant cell transformation.

Emerg Infect Dis, 1997 Apr-Jun, 3(2), 213 - 21
Brucellosis: an overview; Corbel MJ; Brucellosis remains a major zoonosis worldwide . Although many countries have eradicated Brucella abortus from cattle, in some areas Brucella melitensis has emerged as a cause of infection in this species as well as in sheep and goats . Despite vaccination campaigns with the Rev 1 strain, B . melitensis remains the principal cause of human brucellosis . Brucella suis is also emerging as an agent of infection in cattle, thus extending its opportunities to infect humans . The recent isolation of distinctive strains of Brucella from marine mammals has extended its ecologic range . Molecular genetic studies have demonstrated phylogenetic affiliation to Agrobacterium, Phyllobacterium, Ochrobactrum, and Rhizobium . Polymerase chain reaction and gene probe development may provide more effective typing methods . Pathogenicity is related to production of lipopolysaccharides containing a poly N-formyl perosamine O chain, CuZn superoxide dismutase, erythrlose phosphate dehydrogenase, stress-induced proteins related to intracellular survival, and adenine and guanine monophosphate inhibitors of phagocyte functions . Protective immunity is conferred by antibody to lipopolysaccharide and T-cell-mediated macrophage activation triggered by protein antigens . Diagnosis still centers on isolation of the organism and serologic test results, especially enzyme immunoassay, which is replacing other methods . Polymerase chain reaction is also under evaluation . Therapy is based on tetracyclines with or without rifampicin, aminoglycosides, or quinolones . No satisfactory vaccines against human brucellosis are available, although attenuated purE mutants appear promising.

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