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: geneti