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Sample records for agrobacterium rhizogenes atcc

  1. Draft Genome Sequence of Rhizobium rhizogenes Strain ATCC 15834

    PubMed Central

    Kajala, Kaisa; Coil, David A.

    2014-01-01

    Here, we present the draft genome of Rhizobium rhizogenes strain ATCC 15834. The genome contains 7,070,307 bp in 43 scaffolds. R. rhizogenes, also known as Agrobacterium rhizogenes, is a plant pathogen that causes hairy root disease. This hairy root induction has been used in biotechnology for the generation of transgenic root cultures. PMID:25359916

  2. Generation of composite plants using Agrobacterium rhizogenes.

    PubMed

    Taylor, Christopher G; Fuchs, Beth; Collier, Ray; Lutke, W Kevin

    2006-01-01

    Limitations in transformation capability can be a significant barrier in making advances in our understanding of gene function through the use of transgenics. To this end we have developed both tissue culture and non-tissue culture-based methodologies for the production of transgenic roots on wild-type shoots (composite plants). Composite plants are generated by inoculating wild-type shoots with Agrobacterium rhizogenes, which subsequently induces the formation of transgenic roots. The composite plant system allows for "in root" testing of transgenes in the context of a complete plant and can be analyzed in a variety of gene function analyses and plant-microbe interaction studies. In this chapter we provide a tissue culture-based composite plant generation system for Arabidopsis and a non-tissue culture based-method for producing composite plants on a variety of dicotyledonous plant species. Composite plants generated using these methods can be treated like "normal plants," planted in soil and grown in greenhouses or in growth chambers. These methods have been shown to work efficiently for many different species of plants including several that are recalcitrant to transformation.

  3. Proposal for rejection of Agrobacterium tumefaciens and revised descriptions for the genus Agrobacterium and for Agrobacterium radiobacter and Agrobacterium rhizogenes.

    PubMed

    Sawada, H; Ieki, H; Oyaizu, H; Matsumoto, S

    1993-10-01

    The 16S rRNA sequences of seven representative Agrobacterium strains, eight representative Rhizobium strains, and the type strains of Azorhizobium caulinodans and Bradyrhizobium japonicum were determined. These strains included the type strains of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium radiobacter, Agrobacterium vitis, Agrobacterium rubi, Rhizobium fredii, Rhizobium galegae, Rhizobium huakuii, Rhizobium leguminosarum, Rhizobium loti, Rhizobium meliloti, and Rhizobium tropici. A phylogenetic analysis showed that the 15 strains of Agrobacterium and Rhizobium species formed a compact phylogenetic cluster clearly separated from the other members of the alpha subclass of the Proteobacteria. However, Agrobacterium species and Rhizobium species are phylogenetically entwined with one another, and the two genera cannot be separated. In the Agrobacterium species, the strains of biovar 1, biovar 2, Agrobacterium rubi, and Agrobacterium vitis were clearly separated. The two biovars exhibited homogeneity in their phenotypic, chemotaxonomic, and phylogenetic characteristics, and two species should be established for the two biovars. We considered the nomenclature of the two biovars, and revised descriptions of Agrobacterium radiobacter (for the biovar 1 strains) and Agrobacterium rhizogenes (for the biovar 2 strains) are proposed. The name Agrobacterium tumefaciens is rejected because the type strain of this species was assigned to Agrobacterium radiobacter, and consequently the description of the genus Agrobacterium is revised.

  4. Draft Genome Sequence of Agrobacterium rhizogenes Strain NCPPB2659

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work reports the draft genome of Agrobacterium rhizogenes strain NCPPB2659 (also known as strain K599). The assembled genome contains 5,277,347 bp, and is composed of 1 circular chromosome, the Ri virulence plasmid, and 17 scaffolds pertaining to the linear chromosome. The wild type strain cau...

  5. Draft Genome Sequence of Agrobacterium rhizogenes Strain NCPPB2659

    PubMed Central

    Valdes Franco, Jose A.; Collier, Ray; Wang, Yi; Huo, Naxin; Gu, Yong

    2016-01-01

    This work reports the draft genome sequence of Agrobacterium rhizogenes strain NCPPB2659 (also known as strain K599). The assembled genome contains 5,277,347 bp, composed of one circular chromosome, the pRi2659 virulence plasmid, and 17 scaffolds pertaining to the linear chromosome. The wild-type strain causes hairy root disease in dicots and has been used to make transgenic hairy root cultures and composite plants (nontransgenic shoots with transgenic roots). Disarmed variants of the strain have been used to produce stable transgenic monocot and dicot plants. PMID:27469966

  6. Draft Genome Sequence of Agrobacterium rhizogenes Strain NCPPB2659.

    PubMed

    Valdes Franco, Jose A; Collier, Ray; Wang, Yi; Huo, Naxin; Gu, Yong; Thilmony, Roger; Thomson, James G

    2016-01-01

    This work reports the draft genome sequence of Agrobacterium rhizogenes strain NCPPB2659 (also known as strain K599). The assembled genome contains 5,277,347 bp, composed of one circular chromosome, the pRi2659 virulence plasmid, and 17 scaffolds pertaining to the linear chromosome. The wild-type strain causes hairy root disease in dicots and has been used to make transgenic hairy root cultures and composite plants (nontransgenic shoots with transgenic roots). Disarmed variants of the strain have been used to produce stable transgenic monocot and dicot plants. PMID:27469966

  7. Enhanced podophyllotoxin production from Agrobacterium rhizogenes transformed cultures of Podophyllum hexandrum.

    PubMed

    Giri, A; Giri, C C; Dhingra, V; Narasu, M L

    2001-01-01

    Podophyllotoxin, a potent chemotherapeutic agent is obtained from Podophyllum hexandrum Royle. Embryos of P. hexandrum were transformed using different strains of Agrobacterium rhizogenes viz. A4, 15834, K599. Transformed nature of the calli was ascertained and the cultures were further maintained as individual clones. HPLG analysis of transformed cultures depicted a three-fold increase in podophyllotoxin content in comparison to controls.

  8. A technique to study Meloidogyne arenaria resistance in Agrobacterium rhizogenes-transformed peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A reliable peanut root transformation system would be useful to study the functions of genes involved in root biology and disease resistance. The objective of this study was to establish an effective protocol to produce composite plants mediated by Agrobacterium rhizogenes transformation. More tha...

  9. [Production of transgenic sugarbeet plants (Beta vulgaris L.) using Agrobacterium rhizogenes].

    PubMed

    Kishchenko, E M; Komarnitskiĭ, I K; Kuchuk, N V

    2005-01-01

    Normal phenotype sugarbeet plants transformed with Agrobacterium rhizogenes were produced using direct regeneration from explants without hairy root phase. Kanamycin resistant plants and Ri-roots carrying the genes of neomycin phosphotransferase II and b-glucuronidase have been obtained. Integration of transgenes into sugarbeet genome was confirmed with GUS-assay and PCR using primers for the introduced genes. PMID:16018172

  10. Efficient transformation of potato (Solanum tuberosum L.) using a binary vector in Agrobacterium rhizogenes.

    PubMed

    Visser, R G; Jacobsen, E; Witholt, B; Feenstra, W J

    1989-10-01

    We transformed three potato (Solanum tuberosum L.) genotypes by using A. rhizogenes or a mixture of A. rhizogenes and A. tumefaciens. Inoculations of potato stem segments were performed with Agrobacterium rhizogenes AM8703 containing two independent plasmids: the wild-type Ri-plasmid, pRI1855, and the binary vector plasmid, pBI121. In mixed inoculation experiments, Agrobacterium rhizogenes LBA1334 (pRI1855) and Agrobacterium tumefaciens AM8706 containing the disarmed Ti-plasmid (pAL4404) and the binary vector plasmid (pBI121) were mixed in a 1∶1 ratio. The T-DNA of the binary vector plasmid pBI121 contained two marker genes encoding neomycin phosphotransferase, which confers resistance to kanamycin, and β-glucuronidase. Both transformation procedures gave rise to hairy roots on potato stem segments within 2 weeks. With both procedures it was possible to obtain transformed hairy roots, able to grow on kanamycin and possessing β-glucuronidase activity, without selection pressure. The efficiency of the A. rhizogenes AM8703 transformation, however, was much higher than that of the "mixed" transformation. Up to 60% of the hairy roots resulting from the former transformation method were kanamycin resistant and possessed β-glucuronidase activity. There was no correlation between the height of the kanamycin resistance and that of the β-glucuronidase activity in a root clone. Hairy roots obtained from a diploid potato genotype turned out to be diploid in 80% of the cases. Transformed potato plants were recovered from Agrobacterium rhizogenes AM8703-induced hairy roots.

  11. Agrobacterium rhizogenes GALLS protein substitutes for Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

    PubMed

    Hodges, Larry D; Cuperus, Josh; Ream, Walt

    2004-05-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2. PMID:15126468

  12. Agrobacterium rhizogenes-Mediated Transformation of the Parasitic Plant Phtheirospermum japonicum

    PubMed Central

    Ishida, Juliane K.; Yoshida, Satoko; Ito, Masaki; Namba, Shigetou; Shirasu, Ken

    2011-01-01

    Background Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood. Methodology/Principal Findings We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6-dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation. Conclusions We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocols described here will allow functional analysis of genes involved in plant parasitism. PMID:21991355

  13. Agrobacterium rhizogenes GALLS protein contains domains for ATP binding, nuclear localization, and type IV secretion.

    PubMed

    Hodges, Larry D; Vergunst, Annette C; Neal-McKinney, Jason; den Dulk-Ras, Amke; Moyer, Deborah M; Hooykaas, Paul J J; Ream, Walt

    2006-12-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2. PMID:17012398

  14. Influences of Agrobacterium rhizogenes strains, plant genotypes, and tissue types on the induction of transgenic hairy roots in Vitis species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Agrobacterium rhizogenes-mediated induction of transgenic hairy roots was previously demonstrated in Vitis vinifera L. and a few other Vitis species. In this study, 13 Vitis species, including V. aestivalis, V. afghanistan, V. champinii, V. doaniana, V. flexuosa, V. labrusca, V. nesbittiana, V. pal...

  15. Agrobacterium rhizogenes vs auxinic induction for in vitro rhizogenesis of Prosopis chilensis and Nothofagus alpina.

    PubMed

    Caro, Luis A; Santecchia, Natalia; Marinangeli, Pablo A; Curvetto, Néstor R; Hernández, Luis F

    2003-12-01

    The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin. PMID:15002748

  16. An efficient protocol for genetic transformation of watercress (Nasturtium officinale) using Agrobacterium rhizogenes.

    PubMed

    Park, Nam Il; Kim, Jae Kwang; Park, Woo Tae; Cho, Jin Woong; Lim, Yong Pyo; Park, Sang Un

    2011-11-01

    Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties. To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01-0.02 μmol/g of DW) and 4-methoxyglucobrassicin (0.06-0.18 μmol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06-0.21 μmol/g of DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive agent (indole-3-carbinol).

  17. Analysis of core genes supports the reclassification of strains Agrobacterium radiobacter K84 and Agrobacterium tumefaciens AKE10 into the species Rhizobium rhizogenes.

    PubMed

    Velázquez, Encarna; Palomo, José Luis; Rivas, Raúl; Guerra, Hilario; Peix, Alvaro; Trujillo, Martha E; García-Benavides, Pablo; Mateos, Pedro F; Wabiko, Hiroetsu; Martínez-Molina, Eustoquio

    2010-08-01

    Some strains of the former genus Agrobacterium have high biotechnological interest and are currently misclassified. Consequently, in this study, the taxonomic status of the non-pathogenic strain Agrobacterium radiobacter K84, used in biological control, and the tumourigenic strain Agrobacterium tumefaciens AKE10, able to regenerate tobacco transgenic plants, was revised. The phylogenetic analysis of the chromosomal genes rrs, atpD and recA showed that they should be reclassified into Rhizobium rhizogenes. The analysis of virulence genes located in the Ti plasmid (pTi) outside T-DNA showed a common phylogenetic origin among strains AKE10, R. rhizogenes 163C and A. tumefaciens (currently R. radiobacter) C58. However, the genes located inside the T-DNA, mainly the 6b gene, of strain AKE10 were phylogenetically close to those of strain 163C but divergent from those of strain C58. Furthermore, the T-DNA of tumourigenic strains from R. rhizogenes conferred on them the ability to regenerate tumour tissue resembling fasciation in tobacco plants. These results showed the existence of a highly mosaic genetic organization in tumourigenic strains of the genus Rhizobium and provided evidence of the involvement of T-DNA from tumourigenic strains of R. rhizogenes in fasciation of Nicotiana leaves. The data further suggested that pathogenic strains of Rhizobium could be good models to analyse bacterial evolution.

  18. The Agrobacterium rhizogenes GALLS gene encodes two secreted proteins required for genetic transformation of plants.

    PubMed

    Hodges, Larry D; Lee, Lan-Ying; McNett, Henry; Gelvin, Stanton B; Ream, Walt

    2009-01-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are related pathogens that cause crown gall and hairy root diseases, which result from integration and expression of bacterial genes in the plant genome. Single-stranded DNA (T strands) and virulence proteins are translocated into plant cells by a type IV secretion system. VirD2 nicks a specific DNA sequence, attaches to the 5' end, and pilots the DNA into plant cells. A. tumefaciens translocates single-stranded DNA-binding protein VirE2 into plant cells where it likely binds T strands and may aid in targeting them into the nucleus. Although some A. rhizogenes strains lack VirE2, they transfer T strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant for tumor formation. Unlike VirE2, full-length GALLS (GALLS-FL) contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. GALLS-FL and VirE2 contain nuclear localization signals (NLS) and secretion signals. Mutations in any of these domains abolish the ability of the GALLS gene to substitute for virE2. Here, we show that the GALLS gene encodes two proteins from one open reading frame: GALLS-FL and a protein comprised of the C-terminal domain, which initiates at an internal in-frame start codon. On some hosts, both GALLS proteins were required to substitute for VirE2. GALLS-FL tagged with yellow fluorescent protein localized to the nucleus of tobacco cells in an NLS-dependent manner. In plant cells, the GALLS proteins interacted with themselves, VirD2, and each other. VirD2 interacted with GALLS-FL and localized inside the nucleus, where its predicted helicase activity may pull T strands into the nucleus. PMID:18952790

  19. Iron-Binding Compounds from Agrobacterium spp.: Biological Control Strain Agrobacterium rhizogenes K84 Produces a Hydroxamate Siderophore

    PubMed Central

    Penyalver, Ramón; Oger, Philippe; López, María M.; Farrand, Stephen K.

    2001-01-01

    Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing

  20. Agrobacterium rhizogenes: Transformed root cultures for the study of polyacetylene metabolism and biosynthesis

    SciTech Connect

    Marchant, Y.Y.

    1988-02-01

    Biologically active polyacetylenes are produced at low levels by the roots of members of the Coreopsidinae subtribe in the Asteraceae. Ten taxa of Coreopsis and Bidens were tranformed with Agrobacterium rhizogenes Strain A/sub 4/ and hairy root cultures established. These cultures grew rapidly and produced the same arrays of polyacetylenes as intact roots. The use of transformed roots for the study of polyacetylene biosynthesis is described in this paper. The engineering of plants with resistance to herbicides is now a practical reality because there are economic, intellectual and environmental incentives for using recombinant DNA technology in crop improvement programs, and because the biochemical and genetic basis for herbicide resistance is a simple trait conferred by a single gene. The transformation of plants with genes conferring resistance to insects or disease is more daunting, however, as biologically active secondary metabolites such as some alkaloids are typically products of multienzyme reactions. Photoactive polyacetylenes are probably plant defense chemicals and they are derived by a sequence of desaturation steps from oleic acid, which occurs ubiquitously in higher plants. Although the acetylene pathway may encompass as many genetic messages as those for morphine biosynthesis, it is likley that the genes controlling the biosynthesis of polyacetylenes may be isolated, identified in the near future and transferred via Agrobacterium to economically important plants susceptible to pathogen attack. 58 refs., 4 figs., 3 tabs.

  1. The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

    PubMed

    Sharifi, Sara; Sattari, Taher Nejad; Zebarjadi, Alireza; Majd, Ahmad; Ghasempour, Hamidreza

    2014-01-01

    We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10-14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g(-1) of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L(-1) naphthaleneacetic acid and 2 mg L(-1) 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots. PMID:24554840

  2. Horticultural characteristics of transgenic tobacco expressing the rolC gene from Agrobacterium rhizogenes

    SciTech Connect

    Scorza, R.; Zimmerman, T.W.; Cordts, J.M.; Footen, K.J. ); Ravelonandro, M. . Station de Pathologie Vegetale)

    1994-09-01

    Wisconsin 38 tobacco (Nicotiana tabacum L.) leaf discs were transformed with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying the rolC gene from A. rhizogenes and NPT II and GUS genes. Shoots that regenerated on kanamycin-containing medium were confirmed as transgenic through GUS assays, polymerase chain reaction (PCR), Southern blot analyses, and transmission of the foreign genes through the sexual cycle. Transgenic plants were as short as half the height of control plants; were earlier flowering by up to 35 days; and had smaller leaves, shorter internodes, smaller seed capsules, fewer seeds, smaller flowers, and reduced pollen viability. The number of seed capsules, leaf number, and specific root length were similar between transgenic and control plants. Transgenic clones varied in the expression of the rolC-induced growth alterations as did the first generation of seedlings from these clones. Such differences suggested the potential for selecting for different levels of expression. Transformation with the rolC gene presents a potentially useful method of genetically modifying horticultural crops, particularly for flowering date, height, and leaf and flower size. Chemical names used: neomycin phosphotransferase (NPTII), [beta]-glucuronidase (GUS).

  3. Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC.

    PubMed

    Brijwal, Latika; Tamta, Sushma

    2015-01-01

    An efficient protocol for hairy root induction in Berberis aristata DC. was established using two different strains of Agrobacterium rhizogenes, MTCC 532 and 2364 from IMTECH (Institute of Microbial Technology), Chandigarh, India. The strain 532 was more effective than strain 2364 in hairy root induction and in vitro grown callus (61.11 ± 1.60 % transformation frequency) was found to be suitable explant in comparison to leaves (42.59 ± 0.92 % transformation frequency) and nodal segments (34.25 ± 0.92 % transformation frequency) of in vitro grown microshoots for hairy root induction. The presence of rol A and rol B genes during amplification confirmed the transgenic nature of hairy roots and transformed callus. Transformation frequency of callus was further enhanced (from 61.11 ± 1.60 % to 72.22 ± 1.60 %; when infection time was 1 h) by using acetosyringone (100 µM) during co-cultivation period (48 h) on semisolid MS (Murashige and Skoog) medium. In conclusion, this study describes the protocol for hairy root induction which could further be useful for the production of berberin and may reduce the overharvesting of this endangered species from its natural habitat.

  4. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

    DOE PAGES

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T.; Vogel, John P.; Thilmony, Roger

    2016-05-24

    In this study, the genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This studymore » demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticurn. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. turnefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.« less

  5. Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum

    PubMed Central

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T.; Vogel, John P.; Thilmony, Roger

    2016-01-01

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation. PMID:27252729

  6. Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum.

    PubMed

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T; Vogel, John P; Thilmony, Roger

    2016-01-01

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation. PMID:27252729

  7. Use of Agrobacterium rhizogenes Strain 18r12v and Paromomycin Selection for Transformation of Brachypodium distachyon and Brachypodium sylvaticum.

    PubMed

    Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T; Vogel, John P; Thilmony, Roger

    2016-01-01

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.

  8. Assessment of the genetic and phenotypic diversity among rhizogenic Agrobacterium biovar 1 strains infecting solanaceous and cucurbit crops.

    PubMed

    Bosmans, Lien; Álvarez-Pérez, Sergio; Moerkens, Rob; Wittemans, Lieve; Van Calenberge, Bart; Kerckhove, Stefan Van; Paeleman, Anneleen; De Mot, René; Rediers, Hans; Lievens, Bart

    2015-08-01

    Rhizogenic Agrobacterium biovar 1 strains have been found to cause extensive root proliferation on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in substantial economic losses. As these agrobacteria live under similar ecological conditions, infecting a limited number of crops, it may be hypothesized that genetic and phenotypic variation among such strains is relatively low. In this study we assessed the phenotypic diversity as well as the phylogenetic and evolutionary relationships of several rhizogenic Agrobacterium biovar 1 strains from cucurbit and solanaceous crops. A collection of 41 isolates was subjected to a number of phenotypic assays and characterized by MLSA targeting four housekeeping genes (16S rRNA gene, recA, rpoB and trpE) and two loci from the root-inducing Ri-plasmid (part of rolB and virD2). Besides phenotypic variation, remarkable genotypic diversity was observed, especially for some chromosomal loci such as trpE. In contrast, genetic diversity was lower for the plasmid-borne loci, indicating that the studied chromosomal housekeeping genes and Ri-plasmid-borne loci might not exhibit the same evolutionary history. Furthermore, phylogenetic and network analyses and several recombination tests suggested that recombination could be contributing in some extent to the evolutionary dynamics of rhizogenic Agrobacterium populations. Finally, a genomospecies-level identification analysis revealed that at least four genomospecies may occur on cucurbit and tomato crops (G1, G3, G8 and G9). Together, this study gives a first glimpse at the genetic and phenotypic diversity within this economically important plant pathogenic bacterium.

  9. Agravitropic behaviour of roots of rapeseed (Brassica napus L.) transformed by Agrobacterium rhizogenes.

    PubMed

    Odegaard, E; Nielsen, K M; Beisvag, T; Evjen, K; Johnsson, A; Rasmussen, O; Iversen, T H

    1997-10-01

    Transgenic hairy roots of Brassica napus (cv. Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots. The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes. The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia. Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected. The transformed nature of the roots was confirmed by Southern blot analyses. The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings. While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground. The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy. At the ultrastructural level no major differences could be detected between the roots studied. There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type. Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.

  10. Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn).

    PubMed

    Thwe, Aye; Valan Arasu, Mariadhas; Li, Xiaohua; Park, Chang Ha; Kim, Sun Ju; Al-Dhabi, Naif Abdullah; Park, Sang Un

    2016-01-01

    The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum 'Hokkai T10' cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as ftpAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3(,) H1, FtF3'H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 μg/mg DW, respectively), cyanidin 3-O-glucoside (800, 750, and 650 μg/g DW, respectively), and cyanidin 3-O-rutinoside (2410, 1530, and 1170 μg/g DW, respectively). A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat.

  11. Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn)

    PubMed Central

    Thwe, Aye; Valan Arasu, Mariadhas; Li, Xiaohua; Park, Chang Ha; Kim, Sun Ju; Al-Dhabi, Naif Abdullah; Park, Sang Un

    2016-01-01

    The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum ‘Hokkai T10’ cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as ftpAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3, H1, FtF3′H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 μg/mg DW, respectively), cyanidin 3-O-glucoside (800, 750, and 650 μg/g DW, respectively), and cyanidin 3-O-rutinoside (2410, 1530, and 1170 μg/g DW, respectively). A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat. PMID:27014239

  12. Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model.

    PubMed

    Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B; Bailey-Serres, Julia; Brady, Siobhan M

    2014-10-01

    Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

  13. Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model1[W][OPEN

    PubMed Central

    Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A.; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B.; Bailey-Serres, Julia; Brady, Siobhan M.

    2014-01-01

    Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032

  14. Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum l., and California poppy, Eschscholzia californica cham., root cultures.

    PubMed

    Park, S U; Facchini, P J

    2000-06-01

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

  15. Agrobacterium tumefaciens and A. rhizogenes use different proteins to transport bacterial DNA into the plant cell nucleus.

    PubMed

    Ream, Walt

    2009-07-01

    Agrobacterium tumefaciens and A. rhizogenes transport single-stranded DNA (ssDNA; T-strands) and virulence proteins into plant cells through a type IV secretion system. DNA transfer initiates when VirD2 nicks border sequences in the tumour-inducing plasmid, attaches to the 5' end, and pilots T-strands into plant cells. Agrobacterium tumefaciens translocates ssDNA-binding protein VirE2 into plant cells where it targets T-strands into the nucleus. Some A. rhizogenes strains lack VirE2 but transfer T-strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant. VirE2 and full-length GALLS (GALLS-FL) contain nuclear localization sequences that target these proteins to the plant cell nucleus. VirE2 binds cooperatively to T-strands allowing it to move ssDNA without ATP hydrolysis. Unlike VirE2, GALLS-FL contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. VirE2 may accumulate in the nucleus and pull T-strands into the nucleus using the force generated by cooperative DNA binding. GALLS-FL accumulates inside the nucleus where its predicted ATP-dependent strand transferase may pull T-strands into the nucleus. These different mechanisms for nuclear import of T-strands may affect the efficiency and quality of transgenic events in plant biotechnology applications. PMID:21255274

  16. A protocol for sonication-assisted Agrobacterium rhizogenes-mediated transformation of haploid and diploid sugar beet (Beta vulgaris L.) explants.

    PubMed

    Klimek-Chodacka, Magdalena; Baranski, Rafal

    2014-01-01

    Hairy root cultures obtained after Agrobacterium rhizogenes-mediated genetic transformation can serve as a model system for studying plant metabolism and physiology, or can be utilized for the production of secondary metabolites. So far no efficient protocol of hairy root development in sugar beet has been publically released. In this work, two A. rhizogenes strains (A4T and LBA1334) carrying a binary vector pBIN-m-gfp5-ER or pCAMBIA1301 possessing gfp and uidA reporter genes were used to transform petiole explants of haploid and diploid sugar beet genotypes. Five treatment combinations of sonicated-assisted Agrobacterium-mediated transformation were compared. Hairy roots appeared on 0% to 54% of explants depending on the treatment combination used. The highest frequency was achieved when explants of a diploid genotype were sonicated for 15 s in the inoculum containing A. rhizogenes of OD600=0.5 and then co-cultured for three days. Using the same treatment combinations the explants of haploid genotypes developed hairy roots with the frequency ranging from 10% to 36%. Transformation efficiency was independent on the bacterial strain used. The results indicate that haploid sugar beet explants are amenable to transformation using A. rhizogenes, and that the efficiency of that process can be increased by applying short ultrasound treatment.

  17. Effect of Agrobacterium rhizogenes and elicitation on the asiaticoside production in cell cultures of Centella asiatica

    PubMed Central

    Ruslan, Komar; Selfitri, Anggrahaeni Dewi; Bulan, Shella A.; Rukayadi, Yaya; Elfahmi

    2012-01-01

    Background: Centella asiatica (L.) Urb. (Apiaceae) is an important medicinal plant, and it has been using to prepare herbal medicines. The compounds responsible for the biological activity of C. asiatica are triterpenoids such as asiaticoside. Asiaticoside is also important as a marker for standardization of C. asiatica. Due to the low content, there is a need to enhance the production of asiaticoside of C. asiatica. The biotechnological approach is one of the methods that can be used to enhance its production. Objectives: This study was designed to enhance the production of asiaticoside from C. asiatica using A. rhizogenes and elicitation experiments. Materials and Methods: Callus cultures were initiated using Murashige and Skoog (MS) medium supplemented with 1.0 mg/L indole-3-acetic acid (IAA) and 1.0 mg/L 6-benzylaminopurin (BAP). All media were supplemented with 4% (w/w) sucrose and solidified with 0.9% agar. Elicitations were done using pectin, methyl jasmonate, and Cu2+ ions. Transformed hairy root cultures were performed using A. rhizogenes. Results: Callus culture of C. asiatica was successfully initiated. Enhancement of the production of asiaticoside in the callus culture by elicitors pectin was up to 31%; methyl jasmonate (50 μM) in cell suspension cultures at day 14 was up to 171% compared to explant and 494% compared to control callus; copper ion (25 μM) at day 21 was up to 144% compared to explant, and 676% compared to control cell suspension cultures. While enhancement by genetic transformation using A. rhizogenes was 166-172% compare to untransformed roots Conclusion: Elicitation and genetically transformed hairy root cultures of C. asiatica produced asiaticoside up to 172% higher than untreated callus. PMID:22701283

  18. Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis.

    PubMed

    Rana, Mohammad M; Han, Zhuo-Xiao; Song, Da-Peng; Liu, Guo-Feng; Li, Da-Xiang; Wan, Xiao-Chun; Karthikeyan, Alagarsamy; Wei, Shu

    2016-07-15

    Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L(-1) sucrose, 0.1 g·L(-1) l-glutamine and 5 g·L(-1) polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L(-1) sucrose). Additionally, the reporter genes β-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties.

  19. Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis.

    PubMed

    Rana, Mohammad M; Han, Zhuo-Xiao; Song, Da-Peng; Liu, Guo-Feng; Li, Da-Xiang; Wan, Xiao-Chun; Karthikeyan, Alagarsamy; Wei, Shu

    2016-01-01

    Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L(-1) sucrose, 0.1 g·L(-1) l-glutamine and 5 g·L(-1) polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L(-1) sucrose). Additionally, the reporter genes β-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties. PMID:27428960

  20. Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis

    PubMed Central

    Rana, Mohammad M.; Han, Zhuo-Xiao; Song, Da-Peng; Liu, Guo-Feng; Li, Da-Xiang; Wan, Xiao-Chun; Karthikeyan, Alagarsamy; Wei, Shu

    2016-01-01

    Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L−1 sucrose, 0.1 g·L−1 l-glutamine and 5 g·L−1 polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L−1 sucrose). Additionally, the reporter genes β-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties. PMID:27428960

  1. Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants.

    PubMed

    Majumdar, Sukanya; Garai, Saraswati; Jha, Sumita

    2011-05-01

    We have developed an efficient transformation system for Bacopa monnieri, an important Indian medicinal plant, using Agrobacterium rhizogenes strains LBA 9402 and A4. Transformed roots induced by strain LBA 9402 spontaneously dedifferentiated to callus while excised roots induced by strain A4 spontaneously showed induction of shoot buds within 10 days. PCR and RT-PCR analysis revealed the presence and expression of the rolAB and rolC genes at the transcription level in pRi A4 transformed cultures indicating that the TL-DNA was integrated retained and expressed in the A4-Ri transformed shoots. Transformed calli showed the presence of rolAB or rol A, TR and ags genes. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes. Growth and biomass accumulation was significantly higher in the transformed shoots (twofold) and roots (fourfold) than in the non-transformed (WT) plants. In pRi A4-transformed plants, the content of bacopasaponin D, bacopasaponin F, bacopaside II and bacopaside V was enhanced significantly as compared to WT plants of similar age while bacoside A3 and bacopasaponin C content was comparable with that of WT plants. Significant increase in content of five bacopa saponins could be detected in pRi 9402-transformed callus cultures. There is an overall stimulatory effect on accumulation of bacopa saponins in transformed plants and cells of B. monnieri establishing the role of endogenous elicitation by Ri T-DNA of A. rhizogenes.

  2. A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis.

    PubMed

    Young, J M; Kuykendall, L D; Martínez-Romero, E; Kerr, A; Sawada, H

    2001-01-01

    Rhizobium, Agrobacterium and Allorhizobium are genera within the bacterial family Rhizobiaceae, together with Sinorhizobium. The species of Agrobacterium, Agrobacterium tumefaciens (syn. Agrobacterium radiobacter), Agrobacterium rhizogenes, Agrobacterium rubi and Agrobacterium vitis, together with Allorhizobium undicola, form a monophyletic group with all Rhizobium species, based on comparative 16S rDNA analyses. Agrobacterium is an artificial genus comprising plant-pathogenic species. The monophyletic nature of Agrobacterium, Allorhizobium and Rhizobium and their common phenotypic generic circumscription support their amalgamation into a single genus, Rhizobium. Agrobacterium tumefaciens was conserved as the type species of Agrobacterium, but the epithet radiobacter would take precedence as Rhizobium radiobacter in the revised genus. The proposed new combinations are Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium undicola and Rhizobium vitis.

  3. Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation.

    PubMed

    Chattopadhyay, Tirthartha; Roy, Sheuli; Mitra, Adinpunya; Maiti, Mrinal K

    2011-04-01

    Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.

  4. Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes.

    PubMed

    Aarrouf, J; Castro-Quezada, P; Mallard, S; Caromel, B; Lizzi, Y; Lefebvre, V

    2012-02-01

    Pepper is known to be a recalcitrant species to genetic transformation via Agrobacterium tumefaciens. A. rhizogenes-mediated transformation offers an alternative and rapid possibility to study gene functions in roots. In our study, we developed a new and efficient system for A. rhizogenes transformation of the cultivated species Capsicum annuum. Hypocotyls and foliar organs (true leaves and cotyledons) of Yolo Wonder (YW) and Criollo de Morelos 334 (CM334) pepper cultivars were inoculated with the two constructs pBIN-gus and pHKN29-gfp of A. rhizogenes strain A4RS. Foliar explants of both pepper genotypes infected by A4RS-pBIN-gus or A4RS-pHKN29-gfp produced transformed roots. Optimal results were obtained using the combination of the foliar explants with A4RS-pHKN29-gfp. 20.5% of YW foliar explants and 14.6% of CM334 foliar explants inoculated with A4RS-pHKN29-gfp produced at least one root expressing uniform green fluorescent protein. We confirmed by polymerase chain reaction the presence of the rolB and gfp genes in the co-transformed roots ensuring that they integrated both the T-DNA from the Ri plasmid and the reporter gene. We also demonstrated that co-transformed roots of YW and CM334 displayed the same resistance response to Phytophthora capsici than the corresponding untransformed roots. Our novel procedure to produce C. annuum hairy roots will thus support the functional analysis of potential resistance genes involved in pepper P. capsici interaction. PMID:22016085

  5. Scale-Up of Agrobacterium rhizogenes-Mediated Hairy Root Cultures of Rauwolfia serpentina: A Persuasive Approach for Stable Reserpine Production.

    PubMed

    Mehrotra, Shakti; Srivastava, Vikas; Goel, Manoj K; Kukreja, Arun K

    2016-01-01

    Roots of Rauwolfia serpentina, also known as "Sarpagandha" possess high pharmaceutical value due to the presence of reserpine and other medicinally important terpene indole alkaloids. Ever increasing commercial demand of R. serpentina roots is the major reason behind the unsystematic harvesting and fast decline of the species from its natural environment. Considering Agrobacterium rhizogenes-mediated hairy root cultures as an alternative source for the production of plant-based secondary metabolites, the present optimized protocol offers a commercially feasible method for the production of reserpine, the most potent alkaloid from R. serpentina roots. This end-to-end protocol presents the establishment of hairy root culture from the leaf explants of R. serpentina through the infection of A. rhizogenes strain A4 in liquid B5 culture medium and its up-scaling in a 5 L bench top, mechanically agitated bioreactor. The transformed nature of roots was confirmed through PCR-based rol A gene amplification in genomic DNA of putative hairy roots. The extraction and quantification of reserpine in bioreactor grown roots has been done using monolithic reverse phase high-performance liquid chromatography (HPLC). PMID:27108322

  6. Transformation of Althaea officinalis L. by Agrobacterium rhizogenes for the production of transgenic roots expressing the anti-HIV microbicide cyanovirin-N.

    PubMed

    Drake, Pascal M W; de Moraes Madeira, Luisa; Szeto, Tim H; Ma, Julian K-C

    2013-12-01

    The marshmallow plant (Althaea officinalis L.) has been used for centuries in medicine and other applications. Valuable secondary metabolites have previously been identified in Agrobacterium rhizogenes-generated transgenic 'hairy' roots in this species. In the present study, transgenic roots were produced in A. officinalis using A. rhizogenes. In addition to wild-type lines, roots expressing the anti-human immunodeficiency virus microbicide candidate, cyanovirin-N (CV-N), were generated. Wild-type and CV-N root lines were transferred to liquid culture and increased in mass by 49 and 19 % respectively over a 7 day culture period. In the latter, the concentration of CV-N present in the root tissue was 2.4 μg/g fresh weight, with an average secretion rate into the growth medium of 0.02 μg/ml/24 h. A. officinalis transgenic roots may therefore in the future be used not only as a source of therapeutic secondary metabolites, but also as an expression system for the production of recombinant pharmaceuticals.

  7. Expression of the Agrobacterium rhizogenes rolC Gene in a Deciduous Forest Tree Alters Growth and Development and Leads to Stem Fasciation.

    PubMed Central

    Nilsson, O.; Moritz, T.; Sundberg, B.; Sandberg, G.; Olsson, O.

    1996-01-01

    We have altered the growth and development of a deciduous forest tree by transforming hybrid aspen (Populus tremula x Populus tremuloides) with the Agrobacterium rhizogenes rolC gene expressed under the strong cauliflower mosaic virus 35S promoter. We demonstrate that the genetically manipulated perennial plants, after a period of dormancy, maintain the induced phenotypical changes during the second growing period. Furthermore, mass-spectrometrical quantifications of the free and conjugated forms of indole-3-acetic acid and cytokinins and several gibberellins on one transgenic line correlate the induced developmental alterations such as stem fasciation to changes in plant hormone metabolism. We also show that the presence of the RolC protein increases the levels of the free cytokinins, but not by a process involving hydrolysis of the inactive cytokinin conjugates. PMID:12226405

  8. Expression of the Agrobacterium rhizogenes rolC Gene in a Deciduous Forest Tree Alters Growth and Development and Leads to Stem Fasciation.

    PubMed

    Nilsson, O.; Moritz, T.; Sundberg, B.; Sandberg, G.; Olsson, O.

    1996-10-01

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

  9. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brac...

  10. Cell-autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assay for transposon excision in transgenic plants.

    PubMed Central

    Spena, A; Aalen, R B; Schulze, S C

    1989-01-01

    We describe a genetic switch based on the Ac transposable element of maize and the rolC gene of Agrobacterium rhizogenes, a dominant gene, which has pleiotropic effects on plant growth and morphology. Moreover, rolC gene expression under the control of the 35S cauliflower mosaic virus promoter decreases chlorophyll content in transgenic tobacco plants. Chlorophyll is a visible cell-autonomous marker, and it is shown here that the reduction in chlorophyll content caused by the rolC gene product allows us to monitor, in palisade or spongy mesophyll cells, Ac excision events resulting in rolC gene expression as pale-green sectors and spots. Our results indicate that the rolC gene product behaves in a cell-autonomous manner during leaf development, at least as far as chlorophyll accumulation is concerned. In addition, the rolC gene can be useful to evaluate visually if and when a transposable element is active. Most important, we propose the use of a transposable element as a tool to activate expression of morphogenetic genes in a clonal population of cells. This could be particularly useful when studying genes affecting growth and development whose constitutive expression can severely impair regeneration of transgenic plants. PMID:2562512

  11. alpha-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes.

    PubMed

    Burtin, D; Martin-Tanguy, J; Tepfer, D

    1991-02-01

    alpha-dl-Difluoromethylarginine (DFMA) and alpha-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway.

  12. α-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes

    PubMed Central

    Burtin, D.; Martin-Tanguy, J.; Tepfer, D.

    1991-01-01

    α-dl-Difluoromethylarginine (DFMA) and α-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway. Images Figure 1 PMID:16668006

  13. Metabolic engineering of Agrobacterium sp. ATCC31749 for curdlan production from cellobiose.

    PubMed

    Shin, Hyun-Dong; Liu, Long; Kim, Mi-Kyoung; Park, Yong-Il; Chen, Rachel

    2016-09-01

    Curdlan is a commercial polysaccharide made by fermentation of Agrobacterium sp. Its anticipated expansion to larger volume markets demands improvement in its production efficiency. Metabolic engineering for strain improvement has so far been limited due to the lack of genetic tools. This research aimed to identify strong promoters and to engineer a strain that converts cellobiose efficiently to curdlan. Three strong promoters were identified and were used to install an energy-efficient cellobiose phosphorolysis mechanism in a curdlan-producing strain. The engineered strains were shown with enhanced ability to utilize cellobiose, resulting in a 2.5-fold increase in titer. The availability of metabolically engineered strain capable of producing β-glucan from cellobiose paves the way for its production from cellulose. The identified native promoters from Agrobacterium open up opportunities for further metabolic engineering for improved production of curdlan and other products. The success shown here marks the first such metabolic engineering effort in this microbe. PMID:27387419

  14. Production and downstream processing of (1→3)-β-D-glucan from mutant strain of Agrobacterium sp. ATCC 31750

    PubMed Central

    2012-01-01

    We isolated a mutant that produced higher levels of curdlan than the wild strain Agrobacterium sp. ATCC 31750 by chemical mutagenesis using N-methyl-N-nitro-nitrosoguanidine. The mutant strain produced 66 g/L of curdlan in 120 h with a yield of (0.88) while, the wild strain produced 41 g/L in 120 h with a yield of (0.62) in a stirred bioreactor. The mutant could not produce curdlan when the pH was shifted from 7.0 to 5.5 after nitrogen depletion as followed for wild strain. In contrast, pH optimum for cell growth and curdlan production for mutant was found to be 7.0. We optimized the downstream processing of curdlan by varying different volumes of NaOH and HCl for extraction and precipitation of curdlan. The molecular weight of the purified curdlan from the wild and mutant strain was 6.6 × 105 Da and 5.8 × 105 Da respectively. The monosaccharide analyses confirm that curdlan from both wild and mutant strain contains only glucose units. From the NMR and FTIR data, it has been confirmed that curdlan was exclusively composed of β (1 → 3)-D-glucan residues. PMID:22681895

  15. Assessing the Genetic Diversity of Agrobacterium Tumefaciens in CA Walnut Growing Regions and Resistance to the Biocontrol Agent, A. Rhizogenes K84

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crown gall of walnut (Juglans sp.), caused by the bacterium Agrobacterium tumefaciens, greatly impacts the CA walnut industry. To determine the genetic diversity of A. tumefaciens throughout the Central Valley of CA, we collected isolates from ten walnut growing counties. A total of 340 A. tumefac...

  16. In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss.

    PubMed

    Sharafi, Ali; Sohi, Haleh Hashemi; Mirzaee, Hooman; Azadi, Pejman

    2014-10-01

    In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis.

  17. Hairy root induction of Papaver somniferum var. album, a difficult-to-transform plant, by A rhizogenes LBA 9402.

    PubMed

    Le Flem-Bonhomme, V; Laurain-Mattar, D; Fliniaux, M A

    2004-03-01

    Two strains of Agrobacterium rhizogenes (15834, LBA 9402) and one Agrobacterium tumefaciens strain [GV 3101 (PMP90RK, p35SGUS-2)] and four culture media were tested and compared for their ability to induce hairy root formation on wounded Papaver somniferum L. hypocotyls. Five weeks after the infection with A. rhizogenes LBA 9402, hairy roots appeared on 80% of the hypocotyls maintained in the hormone-free liquid medium. Six hairy-root cultures were established. Transformation was confirmed by polymerase chain reaction analysis. One clone was analysed for its alkaloid production. The total alkaloid content was higher in the transformed roots (0.46+/-0.06% DW) than in the untransformed roots (0.32+/-0.05% DW). The transformed roots accumulated three times more codeine (0.18+/-0.02% DW) than intact roots (0.05+/-0% DW). Moreover, morphine (0.255+/-0.03% DW) and sanguinarine (0.014+/-0% DW) were found in the liquid culture medium.

  18. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Agrobacterium rhizogenes and A. tumefaciens are plant pathogenic bacteria causing abnormal tissue growth such as hairy root and crown gall diseases respectively, through the transfer of DNA fragments (T-DNA) bearing functional genes into the host plant genome. This naturally occurring mechanism of g...

  19. Agrobacterium-mediated transformation in Alpinia galanga (Linn.) Willd. for enhanced acetoxychavicol acetate production.

    PubMed

    Rao, Kiranmayee; Chodisetti, Bhuvaneswari; Mangamoori, Lakshmi Narasu; Giri, Archana

    2012-09-01

    Agrobacterium-mediated transformations ensure elevated amounts of secondary metabolite accumulation with genetic and biosynthetic stability. In the present study, Alpinia galanga rich in bioactive compounds was genetically transformed using different strains of Agrobacterium rhizogenes viz. LBA 9402, A(4), 532, 2364 and PRTGus. Even though a higher growth rate was obtained with the LBA 9402 strain, maximum acetoxychavicol acetate accumulation (ACA) was seen in the PRTGus transformant. PRTGus root line has shown 10.1 fold higher ACA content in comparison to the control roots. The lowest ACA production was shown by the A(4) transformant (4.9 fold). The quantification of ACA in the transformed roots was carried out by using HPLC, which was found to be in the order of PRTGus > LBA 9402 > 2364 > 532 > A(4). The fast growth rate of hairy roots, genetic stability and their ability to synthesize more than one metabolite offer a promising system for the production of valuable secondary metabolites.

  20. Agrobacterium virulence gene induction.

    PubMed

    Gelvin, Stanton B

    2006-01-01

    The ability of Agrobacterium to transform plants and other organisms is under highly regulated genetic control. Two Virulence (Vir) proteins, VirA and VirG, function as a two-component regulatory system to sense particular phenolic compounds synthesized by wounded plant tissues. Induction by these phenolic compounds, in the presence of certain neutral or acid sugars, results in activation of other vir genes, leading to the processing of T-DNA from the Ti-plasmid and transfer of T-DNA to recipient host cells. Many plant, and most nonplant, species do not provide sufficient quantities of the correct phenolic compounds to permit efficient Agrobacterium-mediated genetic transformation to occur. In order to transform these species, phenolic inducing compounds must be added to agrobacteria before and/or during cocultivation of recipient cells with the bacteria. This chapter discusses conditions for efficient induction of Agrobacterium virulence genes by phenolic compounds. PMID:16988335

  1. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-01-01

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  2. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-30

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  3. Agrobacterium: nature's genetic engineer.

    PubMed

    Nester, Eugene W

    2014-01-01

    Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun's old observations and also explain why Agrobacterium is nature's genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering. PMID:25610442

  4. Genotypic Variability of Soybean Response to Agrobacterium Strains Harboring the Ti or Ri Plasmids

    PubMed Central

    Owens, Lowell D.; Cress, Dean E.

    1985-01-01

    Twenty four diverse cultivars of soybean (Glycine max [L.] Merrill) and three lines of its annual wild progenitor Glycine soja Sieb and Zucc. were tested for their response to Agrobacterium strains harboring either the Ti (tumor-inducing) plasmid (pTi) from Agrobacterium tumefaciens or the Ri (root-inducing) plasmid (pRi) from Agrobacterium rhizogenes following uniform wounding and inoculation. Based upon gall weight at 8 weeks postinfection, three G. max cultivars (Biloxi, Jupiter, and Peking) and one G. soja line, Plant Introduction (PI) 398.693B, were judged highly susceptible to A. tumefaciens strain A348 (pTiA6), ten genotypes moderately susceptible, 11 weakly susceptible, and two nonsusceptible. Of 26 genotypes inoculated with strain R1000 (pRiA4b), only seven responded in a clearly susceptible fashion by forming small, fleshy roots at internodal infection sites. Cotyledons excised from 1- or 3-day old seedlings of Peking and Biloxi cultivars also formed galls when infected in vitro with agrobacteria carrying either the Ti or Ri plasmid. Tumor lines established from cotyledon and stem galls induced by A. tumefaciens A348 (pTiA6) exhibited the T-DNA borne traits of phytohormone-independent growth and octopine synthesis. Additionally, DNA isolated from cultured tumors hybridized with labeled T-DNA probe. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:16664035

  5. Isozyme gene expression in potato tumors incited by Agrobacterium.

    PubMed

    Oliver, J L

    1986-06-01

    Two plant tumors (crown galls and hairy roots) were experimentally provoked on potato cv. 'Désirée' by oncogenic strains of Agrobacterium tumefaciens and A. rhizogenes. A marked shift in the expression of some organ-specific genes occurred in crown galls derived from the central zone of tubers: two novel isozyme genes (Est-B and Pox-E) were expressed, two others (Est-C and Pox-F) were suppressed and the remaining ones were maintained in the original state. When the starting tissue was the stem segment, a smaller shift occurred, namely the activation of Adh-A and the suppression of Pox-F. In all cases, the isozyme profiles characterizing all crown galls, whatever their origin, were identical. Under normal aeration conditions, Adh-A was not expressed in either tumoral or non-tumoral roots. However, under the relative anaerobic conditions of in vitro cultures, a difference existed between both types of roots: Adh-A was expressed in normal but not in tumoral roots. This means that hairy roots can tolerate higher levels of anaerobiosis without giving rise to an anaerobic response. For the remaining isozymes, no alteration occurred in either organized (hairy root) or unorganized (crown gall) tumors, as compared to the corresponding non-tumoral tissues (normal root and callus, respectively). PMID:24247945

  6. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop.

    PubMed

    Kyndt, Tina; Quispe, Dora; Zhai, Hong; Jarret, Robert; Ghislain, Marc; Liu, Qingchang; Gheysen, Godelieve; Kreuze, Jan F

    2015-05-01

    Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world's arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.

  7. The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop

    PubMed Central

    Kyndt, Tina; Quispe, Dora; Zhai, Hong; Jarret, Robert; Ghislain, Marc; Liu, Qingchang; Gheysen, Godelieve

    2015-01-01

    Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world’s arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops. PMID:25902487

  8. Endophthalmitis caused by Agrobacterium radiobacter.

    PubMed

    Pierre-Filho, Paulo de Tarso P; Ribeiro, Ana Paula Y; Passos, Elane D; Torigoe, Marcelo; de Vasconcellos, José Paulo C

    2003-01-01

    Infections due to Agrobacterium radiobacter are rare. This study reports 2 cases of A. radiobacter endophthalmitis. To the authors' knowledge, these are only the second and third reported cases of endophthalmitis caused by this Gram-negative rod.

  9. Phenotypic analyses of Agrobacterium.

    PubMed

    Morton, Elise R; Fuqua, Clay

    2012-05-01

    Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for simple phenotypic characterizations of A. tumefaciens. PMID:22549164

  10. Laboratory maintenance of Agrobacterium.

    PubMed

    Morton, Elise R; Fuqua, Clay

    2012-02-01

    Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis, virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool, and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for laboratory cultivation of A. tumefaciens. PMID:22307549

  11. Genetic manipulation of Agrobacterium.

    PubMed

    Morton, Elise R; Fuqua, Clay

    2012-05-01

    Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens, this is called the Ti (tumor-inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for genetic manipulation of A. tumefaciens. PMID:22549163

  12. Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.

    PubMed

    Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S; Mirza, Bushra

    2014-01-01

    Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens. PMID:24817272

  13. Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.

    PubMed

    Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S; Mirza, Bushra

    2014-01-01

    Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.

  14. Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid.

    PubMed

    Ramírez-Bahena, Martha H; Vial, Ludovic; Lassalle, Florent; Diel, Benjamin; Chapulliot, David; Daubin, Vincent; Nesme, Xavier; Muller, Daniel

    2014-04-01

    Linear chromosomes are atypical in bacteria and likely a secondary trait derived from ancestral circular molecules. Within the Rhizobiaceae family, whose genome contains at least two chromosomes, a particularity of Agrobacterium fabrum (formerly A. tumefaciens) secondary chromosome (chromid) is to be linear and hairpin-ended thanks to the TelA protelomerase. Linear topology and telA distributions within this bacterial family was screened by pulse field gel electrophoresis and PCR. In A. rubi, A. larrymoorei, Rhizobium skierniewicense, A. viscosum, Agrobacterium sp. NCPPB 1650, and every genomospecies of the biovar 1/A. tumefaciens species complex (including R. pusense, A. radiobacter, A. fabrum, R. nepotum plus seven other unnamed genomospecies), linear chromid topologies were retrieved concomitantly with telA presence, whereas the remote species A. vitis, Allorhizobium undicola, Rhizobium rhizogenes and Ensifer meliloti harbored a circular chromid as well as no telA gene. Moreover, the telA phylogeny is congruent with that of recA used as a marker gene of the Agrobacterium phylogeny. Collectively, these findings strongly suggest that single acquisition of telA by an ancestor was the founding event of a large and diverse clade characterized by the presence of a linear chromid. This clade, characterized by unusual genome architecture, appears to be a relevant candidate to serve as a basis for a possible redefinition of the controversial Agrobacterium genus. In this respect, investigating telA in sequenced genomes allows to both ascertain the place of concerned strains into Agrobacterium spp. and their actual assignation to species/genomospecies in this genus.

  15. Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens.

    PubMed

    Ye, Xudong; Williams, Edward J; Shen, Junjiang; Johnson, Susan; Lowe, Brenda; Radke, Sharon; Strickland, Steve; Esser, James A; Petersen, Michael W; Gilbertson, Larry A

    2011-08-01

    Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation.

  16. Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation.

    PubMed

    Swain, S S; Sahu, L; Pal, A; Barik, D P; Pradhan, C; Chand, P K

    2012-02-01

    Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 μM) to an OD(660) ≅ 0.6, diluted to a density of 10(9) cells ml(-1), followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 μg ml(-1)). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T ( L )-DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T ( R )-DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation. PMID:22806869

  17. Barley Transformation Using Agrobacterium-Mediated Techniques

    NASA Astrophysics Data System (ADS)

    Harwood, Wendy A.; Bartlett, Joanne G.; Alves, Silvia C.; Perry, Matthew; Smedley, Mark A.; Leyland, Nicola; Snape, John W.

    Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.

  18. Agrobacterium and Tumor Induction: A Model System.

    ERIC Educational Resources Information Center

    Lennox, John E.

    1980-01-01

    The author offers laboratory procedures for experiments using the bacterium, Agrobacterium tumefaciens, which causes crown gall disease in a large number of plants. Three different approaches to growing a culture are given. (SA)

  19. Agrobacterium-mediated transformation of cotton.

    PubMed

    Zhang, Baohong

    2013-01-01

    There are many methods and techniques that can be used to transfer foreign genes into cells. In plant biotechnology, Agrobacterium-mediated transformation is a widely used traditional method for inserting foreign genes into plant genome and obtaining transgenic plants, particularly for dicot plant species. Agrobacterium-mediated transformation of cotton involves several important and also critical steps, which includes coculture of cotton explants with Agrobacterium, induction and selection of stable transgenic cell lines, recovery of plants from transgenic cells majorly through somatic embryogenesis, and detection and expression analysis of transgenic plants. In this chapter, we describe a detailed step-by-step protocol for obtaining transgenic cotton plants via Agrobacterium-mediated transformation.

  20. Proposal that Agrobacterium radiobacter has priority over Agrobacterium tumefaciens. Request for an opinion.

    PubMed

    Young, J M; Pennycook, S R; Watson, D R W

    2006-02-01

    It is proposed that Agrobacterium radiobacter has priority as the earlier heterotypic (subjective) synonym when it is united with Agrobacterium tumefaciens. The nomenclatural status of A. tumefaciens as a later heterotypic synonym of the united species is not lost and it remains the type species of the genus. Request for an opinion.

  1. Transformation of rice mediated by Agrobacterium tumefaciens.

    PubMed

    Hiei, Y; Komari, T; Kubo, T

    1997-09-01

    Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing 'competent' cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice. PMID:9291974

  2. Genome Sequence of Propionibacterium acidipropionici ATCC 55737.

    PubMed

    Luna-Flores, Carlos H; Nielsen, Lars K; Marcellin, Esteban

    2016-01-01

    Propionibacterium acidipropionici produces propionic acid as its main fermentation product. Traditionally derived from fossil fuels, environmental and sustainable issues have revived the interest in producing propionic acid using biological resources. Here, we present the closed sequence of Propionibacterium acidipropionici ATCC 55737, an efficient propionic acid producer. PMID:27198010

  3. The production of class III plant peroxidases in transgenic callus cultures transformed with the rolB gene of Agrobacterium rhizogenes.

    PubMed

    Shkryl, Y N; Veremeichik, G N; Bulgakov, V P; Avramenko, T V; Günter, E A; Ovodov, Y S; Muzarok, T I; Zhuravlev, Y N

    2013-10-10

    The production of plant peroxidases by plant cell cultures is of great interest because of the potential for industrial applications. We used plant cell cultures overexpressing the rolB gene to produce increased amounts of plant class III peroxidases. The rolB gene ensured the stable and permanent activation of peroxidase activity in the transformed callus cultures of different plants. In particular, the total peroxidase activity in transformed Rubia cordifolia cells was increased 23-86-fold, and the abundance of the major peroxidase gene transcripts was increased 17-125-fold (depending on the level of rolB expression) compared with non-transformed control calli. The peroxidase-activating effect of rolB was greater than that of other peroxidase inducers, such as external stresses and methyl jasmonate.

  4. Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes.

    PubMed

    Wan, Shen; Johnson, Amanda M; Altosaar, Illimar

    2012-02-01

    The nitrous oxide (N(2)O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N(2)O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N(2)OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N(2)OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N(2)OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 µmol N(2)O reduced min(-1) g(-1) root protein. Another event, plant line 1.9, also demonstrated high specific activity of N(2)OR, 13.2 µmol N(2)O reduced min(-1) g(-1) root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N(2)O that has continued to increase linearly (about 0.26% year(-1)) over the past half-century. PMID:22423324

  5. Enhanced morphinan alkaloid production in hairy root cultures of Papaver bracteatum by over-expression of salutaridinol 7-o-acetyltransferase gene via Agrobacterium rhizogenes mediated transformation.

    PubMed

    Sharafi, Ali; Hashemi Sohi, Haleh; Mousavi, Amir; Azadi, Pejman; Dehsara, Bahareh; Hosseini Khalifani, Bahman

    2013-11-01

    Papaver bracteatum is an important medicinal plant valued for its high content of thebaine and an alternative to P. somniferum for benzylisoquinoline alkaloid production. Salutaridinol 7-o-acetyltransferase (SalAT) is a key gene in morphinan alkaloids biosynthesis pathway. Over expression of SalAT gene was used for metabolic engineering in P. bracteatum hairy root cultures. Transcript level of the salutaridinol 7-o-acetyltransferase gene in transgenic hairy root lines increased up to 154 and 128 % in comparison with hairy roots without SalAT over expression and wild type roots, respectively. High performance liquid chromatography analysis showed that the transgenic hairy roots relatively improved levels of thebaine (1.28 % dry weight), codeine (0.02 % dry weight) and morphine (0.03 % dry weight) compared to those hairy roots without SalAT over expression. This suggests that P. bracteatum hairy roots expressing the SalAT gene could be potentially used for the production of valuable morphinan alkaloids.

  6. Establishment of Hairy Root Cultures by Agrobacterium Rhizogenes Mediated Transformation of Isatis Tinctoria L. for the Efficient Production of Flavonoids and Evaluation of Antioxidant Activities

    PubMed Central

    Luo, Meng; Wei, Zuo-Fu; Zu, Yuan-Gang; Ma, Wei; Fu, Yu-Jie

    2015-01-01

    In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 μg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC50 values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs. PMID:25785699

  7. Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes.

    PubMed

    Wan, Shen; Johnson, Amanda M; Altosaar, Illimar

    2012-02-01

    The nitrous oxide (N(2)O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N(2)O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N(2)OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N(2)OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N(2)OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 µmol N(2)O reduced min(-1) g(-1) root protein. Another event, plant line 1.9, also demonstrated high specific activity of N(2)OR, 13.2 µmol N(2)O reduced min(-1) g(-1) root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N(2)O that has continued to increase linearly (about 0.26% year(-1)) over the past half-century.

  8. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    PubMed Central

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium. PMID:25520733

  9. Draft Genome Sequences of Agrobacterium nepotum Strain 39/7T and Agrobacterium sp. Strain KFB 330.

    PubMed

    Kuzmanović, Nemanja; Puławska, Joanna; Prokić, Anđelka; Ivanović, Milan; Zlatković, Nevena; Gašić, Katarina; Obradović, Aleksa

    2015-01-01

    Tumorigenic strains of Agrobacterium spp. are responsible for crown gall disease of numerous plant species. We present here draft genome sequences of nonpathogenic Agrobacterium nepotum strain 39/7(T) (CFBP 7436(T), LMG 26435(T)), isolated from crown gall tumor on Prunus cerasifera, and tumorigenic Agrobacterium sp. strain KFB 330 (CFBP 8308, LMG 28674), isolated from galls on raspberry. PMID:25908139

  10. Production of triterpenoid anti-cancer compound taraxerol in Agrobacterium-transformed root cultures of butterfly pea (Clitoria ternatea L.).

    PubMed

    Swain, Swasti S; Rout, Kedar K; Chand, Pradeep K

    2012-10-01

    Independent transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) were established using explant co-cultivation with Agrobacterium rhizogenes. Rhizoclones capable of sustained growth were maintained under low illumination in auxin-free agar-solidified MS medium through subcultures at periodic intervals. Integration of T(L)-DNA rolB gene in the transformed rhizoclone genome was verified by Southern blot hybridization, and the transcript expression of T(R)-DNA ags and man2 genes was ascertained by reverse transcription polymerase chain reaction analysis. The major compound isolated and purified from the transformed root extracts was identified as the pentacyclic triterpenoid compound taraxerol using IR, (1)H-NMR, and (13)C-NMR spectroscopy. The taraxerol yield in cultured hairy roots, as quantified by HPTLC analysis, was up to 4-fold on dry weight basis compared to that in natural roots. Scanning of bands from cultured transformed roots and natural roots gave super-imposable spectra with standard taraxerol, suggesting a remarkable homology in composition. To date, this is the first report claiming production of the cancer therapeutic phytochemical taraxerol in genetically transformed root cultures as a viable alternative to in vivo roots of naturally occurring plant species. PMID:22843061

  11. Analysis of Agrobacterium tumefaciens plasmid pTiC58 replication region with a novel high-copy-number derivative.

    PubMed Central

    Gallie, D R; Hagiya, M; Kado, C I

    1985-01-01

    The origin of replication, ori, of the nopaline tumor-inducing plasmid, pTiC58, mapped in a region that shares sequence homology with octopine plasmids pTiAch5 and pTiB6. Within this region, the minimum amount of DNA necessary for maintaining autonomous replication was a 2.6-kilobase region, which also comprised the incompatibility function inc. pTiC58 derivatives containing inc were incompatible with Agrobacterium tumefaciens plasmids pTiC58, pTiD1439, pTiAch5, pTi15955, and pTiA5 and were compatible with A. rhizogenes plasmid pRi12. Situated adjacent to the origin region was a 1.5-kilobase par segment involved in stable inheritance of pTiC58 under nonselective growth conditions. When par was present, plasmid maintenance approached that of the wild-type pTiC58. Rapid loss from the cell population was observed for plasmids not containing this locus. Another 1.5-kilobase region, cop, positively regulated pTiC58 copy number, enabling certain pTiC58 derivatives to exist at a copy number up to 80 times higher than that of wild-type pTiC58. Deletions within the cop locus resulted in reduced copy number. The ori/inc regions were flanked on either side by the par and cop loci. Images PMID:3972769

  12. Agrobacterium-mediated transformation: rice transformation.

    PubMed

    Slamet-Loedin, Inez H; Chadha-Mohanty, Prabhjit; Torrizo, Lina

    2014-01-01

    Agrobacterium is a common soil bacterium with natural capacity for trans-kingdom transfer of genetic information by transferring its T-DNA into the eukaryotic genome. In agricultural plant biotechnology, combination of non-phytopathogenic strain of Agrobacterium tumefaciens with modified T-DNA and vir-genes in a binary vector system is the most widely utilized system for genetic improvement in diverse plant species and for gene function validation. Here we have described a highly efficient A. tumefaciens-mediated transformation system for indica and japonica rice cultivars based on an immature embryo system.

  13. Cloning and nucleotide sequence of the hemA gene of Agrobacterium radiobacter.

    PubMed

    Drolet, M; Sasarman, A

    1991-04-01

    The hemA gene of Agrobacterium radiobacter ATCC4718 was identified by hybridization with a hemA probe from Rhizobium meliloti and cloned by complementation of a hemA mutant of Escherichia coli K12. E. coli hemA transformants carrying the hemA gene of Agrobacterium showed delta-aminolevulinic acid synthetase (delta-ALAS) activity in vitro. The hemA gene was carried on a 4.4 kb EcoRI fragment which could be reduced to a 2.6 kb EcoRI-SstI fragment without affecting its complementing or delta-ALAS activity. The sequence of the hemA gene showed an open reading frame of 1215 nucleotides, which could code for a protein of 44,361 Da. This is very close to the molecular weight of the HemA protein obtained using an in vitro coupled transcription-translation system (45,000 Da). Comparison of amino acid sequences of the delta-ALAS of A. radiobacter and Bradyrhizobium japonicum showed strong homology between the two enzymes; less, but still significant, homology was observed when A. radiobacter and human delta-ALAS were compared. Primer extension experiments enabled us to identify two promoters for the hemA gene of A. radiobacter. One of these promoters shows some similarity to the first promoter of the hemA gene of R. meliloti.

  14. Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production

    PubMed Central

    Elfahmi; Suhandono, Sony; Chahyadi, Agus

    2014-01-01

    Background: Artemisinin, a sesquiterpene lactone endoperoxide isolated from the medicinal plant Artemisia annua L., is a choice and effective drug for malaria treatment. Due to the low yield of artemisinin in plants, there is a need to enhance the production of artemisinin from A. annua and biotechnological technique may be one of the methods that can be used for the purpose. Aim: To study the transformation efficiency of Agrobacterium tumefaciens in A. annua that could be applied to enhance the production of artemisinin by means of transgenic plants. Setting and Designs: The factors influencing Agrobacterium-mediated transformation of A. annua were explored to optimize the transformation system, which included A. tumefaciens strain and effect of organosilicone surfactants. Three strains of A. tumefaciens, that is, LBA4404, GV1301, and AGL1 harboring the binary vector pCAMBIA 1303 have been used for transformation. The evaluation was based on transient β-glucuronidase (GUS). Materials and Methods: Plant cell cultures were inniatiated from the seeds of A. annua using the germination Murashige and Skoog medium. A. tumefaciens harboring pCAMBIA were tranformed into the leaves of A.annua cultures from 2-week-old-seedling and 2-month-old-seedling for 15 min by vacuum infiltration. Transformation efficiency was determinated by measuring of blue area (GUS expression) on the whole leaves explant using ImageJ 1.43 software. Two organosilicon surfactants, that is, Silwet L-77 and Silwet S-408 were used to improve the transformation efficiency. Results: The transformation frequency with AGL1 strain was higher than GV3101 and LBA4404 which were 70.91, 49.25, and 45.45%, respectively. Effect of organosilicone surfactants, that is, Silwet L-77 and Silwet S-408 were tested on A. tumefaciens AGL1 and GV3101 for their level of transient expression, and on A. rhizogenes R1000 for its hairy root induction frequency. For AGL1, Silwet S-408 produced higher level of expression than

  15. Agrobacterium: nature’s genetic engineer

    PubMed Central

    Nester, Eugene W.

    2015-01-01

    Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun’s old observations and also explain why Agrobacterium is nature’s genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering. PMID:25610442

  16. Role for Rhizobium rhizogenes K84 cell envelope polysaccharides in surface interactions.

    PubMed

    Abarca-Grau, Ana M; Burbank, Lindsey P; de Paz, Héctor D; Crespo-Rivas, Juan C; Marco-Noales, Ester; López, María M; Vinardell, Jose M; von Bodman, Susanne B; Penyalver, Ramón

    2012-03-01

    Rhizobium rhizogenes strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a wcbD ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of wcbD in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the rkpK gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The rkpK mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms. PMID:22210213

  17. Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482.

    PubMed

    Wakarchuk, Warren W; Brochu, Denis; Foote, Simon; Robotham, Anna; Saxena, Hirak; Erak, Tamara; Kelly, John

    2016-01-01

    The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.

  18. Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482

    PubMed Central

    Wakarchuk, Warren W.; Brochu, Denis; Foote, Simon; Robotham, Anna; Saxena, Hirak; Erak, Tamara; Kelly, John

    2016-01-01

    The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization. PMID:26950732

  19. VIP1: linking Agrobacterium-mediated transformation to plant immunity?

    PubMed

    Liu, Yukun; Kong, Xiangpei; Pan, Jiaowen; Li, Dequan

    2010-08-01

    Agrobacterium tumefaciens is the most efficient vehicle used today for the production of transgenic plants and plays an essential role in basic scientific research and in agricultural biotechnology. Previously, plant VirE2-interacting protein 1 (VIP1) was shown to play a role in Agrobacterium-mediated transformation. Recent reports demonstrate that VIP1, as one of the bZIP transcription factors, is also involved in plant immunity responses. Agrobacterium is able to activate and abuse VIP1 for transformation. These findings highlight Agrobacterium-host interaction and unveil how Agrobacterium hijacks host cellular mechanism for its own benefit. This review focuses on the roles played by VIP1 in Agrobacterium-mediated transformation and plant immunity. PMID:20473505

  20. VIP1: linking Agrobacterium-mediated transformation to plant immunity?

    PubMed

    Liu, Yukun; Kong, Xiangpei; Pan, Jiaowen; Li, Dequan

    2010-08-01

    Agrobacterium tumefaciens is the most efficient vehicle used today for the production of transgenic plants and plays an essential role in basic scientific research and in agricultural biotechnology. Previously, plant VirE2-interacting protein 1 (VIP1) was shown to play a role in Agrobacterium-mediated transformation. Recent reports demonstrate that VIP1, as one of the bZIP transcription factors, is also involved in plant immunity responses. Agrobacterium is able to activate and abuse VIP1 for transformation. These findings highlight Agrobacterium-host interaction and unveil how Agrobacterium hijacks host cellular mechanism for its own benefit. This review focuses on the roles played by VIP1 in Agrobacterium-mediated transformation and plant immunity.

  1. Transformation of oil palm using Agrobacterium tumefaciens.

    PubMed

    Izawati, Abang Masli Dayang; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul

    2012-01-01

    Transgenic oil palm (Elaeis guineensis Jacq.) plantlets are regenerated after Agrobacterium tumefaciens-mediated transformation of embryogenic calli derived from young leaves of oil palm. The calli are transformed with an Agrobacterium strain, LBA4404, harboring the plasmid pUBA, which carries a selectable marker gene (bar) for resistance to the herbicide Basta and is driven by a maize ubiquitin promoter. Modifications of the transformation method, treatment of the target tissues using acetosyringone, exposure to a plasmolysis medium, and physical injury via biolistics are applied. The main reasons for such modifications are to activate the bacterial virulence system and, subsequently, to increase the transformation efficiency. Transgenic oil palm cells are selected and regenerated on a medium containing herbicide Basta. Molecular analyses revealed the presence and integration of the introduced bar gene into the genome of the transformants. PMID:22351008

  2. Transient Expression of Viral Proteins in Plants Using Agrobacterium tumefaciens.

    PubMed

    Hitzeroth, Inga I; van Zyl, Albertha R

    2016-01-01

    Transient expression of viral proteins in plants is a novel alternative to other expression platforms. The viral proteins can be used as potential vaccines or in diagnostics. Nicotiana benthamiana leaves or whole plants are infiltrated with recombinant Agrobacterium that harbor the gene of interest. Protein expression in the plants is rapid and results are obtained within 2-7 days. Here we describe how to make electrocompetent Agrobacterium, how to transform Agrobacterium, how to infiltrate leaves or plants with the recombinant Agrobacterium, and lastly how to extract the protein for analysis by gel electrophoresis. PMID:27076324

  3. Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.

    PubMed

    Song, Geun Cheol; Lee, Soohyun; Hong, Jaehwa; Choi, Hye Kyung; Hong, Gun Hyong; Bae, Dong-Won; Mysore, Kirankumar S; Park, Yong-Soon; Ryu, Choong-Min

    2015-07-01

    Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium. PMID:25676198

  4. Agrobacterium radiobacter bacteremia in pediatric patients: case report and review.

    PubMed

    Amaya, Rene A; Edwards, Morven S

    2003-02-01

    Agrobacterium radiobacter is an opportunistic pathogen often associated with indwelling catheters. We report five children with central venous catheter-associated A. radiobacter bacteremia and review the characteristics of pediatric Agrobacterium infections. Cure was achieved with appropriate antibiotics, often ticarcillin-clavulanate and gentamicin, and removal of the catheter.

  5. Catheter infection caused by an unusual pathogen, Agrobacterium radiobacter.

    PubMed

    Potvliege, C; Vanhuynegem, L; Hansen, W

    1989-09-01

    The genus Agrobacterium is composed of several phytopathogenic species occurring worldwide in soils. One nontumorigenic species, Agrobacterium radiobacter, has occasionally been isolated from clinical specimens, but its pathogenic role in these cases has been difficult to ascertain since agrobacteria are usually isolated in association with other bacteria. We report the case of a central venous catheter infection and present the characteristics of A. radiobacter.

  6. Isolation of Agrobacterium radiobacter from a central venous catheter.

    PubMed

    Hammerberg, O; Bialkowska-Hobrzanska, H; Gopaul, D

    1991-05-01

    A case of septicemia caused by Agrobacterium radiobacter is reported in a patient undergoing chemotherapy treatment who had recently been neutropenic. Agrobacterium radiobacter was isolated from the Hickman line blood culture. The patient responded favorably to removal of the Hickman catheter and treatment with amikacin and piperacillin. The molecular and biochemical characteristics of the isolate are presented.

  7. Agrobacterium tumefaciens responses to plant-derived signaling molecules

    PubMed Central

    Subramoni, Sujatha; Nathoo, Naeem; Klimov, Eugene; Yuan, Ze-Chun

    2014-01-01

    As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium–plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its Transferred DNA (T-DNA) from its Tumor-inducing (Ti) plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA), cytokinin (CK), and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS) to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including γ-amino butyric acid and salicylic acid (SA) to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays) also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium–plant interactions. PMID:25071805

  8. Agrobacterium-mediated transformation of Fusarium proliferatum.

    PubMed

    Bernardi-Wenzel, J; Quecine, M C; Azevedo, J L; Pamphile, J A

    2016-01-01

    Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process. PMID:27323127

  9. Agrobacterium-mediated transformation of Fusarium proliferatum.

    PubMed

    Bernardi-Wenzel, J; Quecine, M C; Azevedo, J L; Pamphile, J A

    2016-06-03

    Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process.

  10. Draft Genome Sequence of Mycobacterium brumae ATCC 51384

    PubMed Central

    D'Auria, Giuseppe

    2016-01-01

    Here, we report the draft genome sequence of Mycobacterium brumae type strain ATCC 51384. This is the first draft genome sequence of M. brumae, a nonpathogenic, rapidly growing, nonchromogenic mycobacterium, with immunotherapeutic capacities. PMID:27125480

  11. Draft Genome Sequence of Mycobacterium brumae ATCC 51384.

    PubMed

    D'Auria, Giuseppe; Torrents, Eduard; Luquin, Marina; Comas, Iñaki; Julián, Esther

    2016-01-01

    Here, we report the draft genome sequence of Mycobacterium brumae type strain ATCC 51384. This is the first draft genome sequence of M. brumae, a nonpathogenic, rapidly growing, nonchromogenic mycobacterium, with immunotherapeutic capacities. PMID:27125480

  12. Genome sequence of the fish pathogen Flavobacterium columnare ATCC 49512

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare is a Gram-negative, rod shaped, motile, and highly prevalent fish pathogen causing columnaris disease in freshwater fish worldwide. Here, we present the complete genome sequence of F. columnare strain ATCC 49512. ...

  13. Degradation of the ferric chelate of EDTA by a pure culture of an Agrobacterium sp

    SciTech Connect

    Lauff, J.J.; Coogan, L.A.; Breitfeller, J.M. ); Steele, D.B. )

    1990-11-01

    A pure culture of an Agrobacterium sp. (deposited as ATCC 55002) the mineralizes the ferric chelate of EDTA (ferric-EDTA) was isolated by selective enrichment from a treatment facility receiving industrial waste containing ferric-EDTA. The isolated grew on ferric-EDTA as the sole carbon source at concentrations exceeding 100 mM. As the degradation proceeded, carbon dioxide, ammonia, and an unidentified metabolite(s) were produced; the pH increased, and iron was precipitated from solution. The maximum rate of degradation observed with sodium ferric-EDTA as the substrate was 24 mM/day. At a substrate concentration of 35 mM, 90% of the substrate was degraded in 3 days and 70% of the associated chemical oxygen demand was removed from solution. Less than 15% of the carbon initially present was incorporated into the cell mass. Significant growth of this strain was not observed with uncomplexed EDTA as the sole carbon source at comparable concentrations; however, the ferric chelate of propylenediaminetetraacetic acid (ferric-PDTA) did support growth.

  14. Evidence for Agrobacterium-induced apoptosis in maize cells.

    PubMed

    Hansen, G

    2000-06-01

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

  15. Agrobacterium tumefaciens Is a Diazotrophic Bacterium

    PubMed Central

    Kanvinde, Lalita; Sastry, G. R. K.

    1990-01-01

    This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grow on nitrogen-free medium, reduce acetylene to ethylene, and incorporate 15N supplied as 15N2. As with most other well-characterized diazotrophic bacteria, the presence of NH4+ in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship. Images PMID:16348237

  16. Transport of nonmetabolizable opines by Agrobacterium tumefaciens

    SciTech Connect

    Krishnan, M.; Burgner, J.W.; Chilton, W.S.; Gelvin, S.B. )

    1991-01-01

    We have examined the uptake of ({sup 14}C)octopine and ({sup 14}C)nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with ({sup 14}C)octopine and ({sup 14}C)nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid.

  17. Agrobacterium-mediated transformation of Brachypodium distachyon.

    PubMed

    Thole, Vera; Vain, Philippe

    2012-01-01

    Brachypodium distachyon is an attractive genomics and biological model system for grass research. Recently, the complete annotated genome sequence of the diploid line Bd21 has been released. Genetic transformation technologies are critical for the discovery and validation of gene function in Brachypodium. Here, we describe an efficient procedure enabling the Agrobacterium-mediated transformation of a range of diploid and polyploid genotypes of Brachypodium. The procedure relies on the transformation of compact embryogenic calli derived from immature embryos using either chemical selection alone or a combination of chemical and visual screening of transformed tissues and plants. Transformation efficiencies of around 20% can routinely be achieved using this protocol. In the context of the BrachyTAG programme (BrachyTAG.org), this procedure made possible the mass production of Bd21T-DNA mutant plant lines.

  18. Agrobacterium tumefaciens is a diazotrophic bacterium

    SciTech Connect

    Kanvinde, L.; Sastry, G.R.K. )

    1990-07-01

    This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grown on nitrogen-free medium, reduce acetylene to ethylene, and incorporate {sup 15}N supplied as {sup 15}N{sub 2}. As with most other well-characterized diazotrophic bacteria, the presence of NH{sub 4}{sup +} in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship.

  19. Attachment of Agrobacterium to plant surfaces

    PubMed Central

    Matthysse, Ann G.

    2014-01-01

    Agrobacterium tumefaciens binds to the surfaces of inanimate objects, plants, and fungi. These bacteria are excellent colonizers of root surfaces. In addition, they also bind to soil particles and to the surface of artificial or man-made substances, such as polyesters and plastics. The mechanisms of attachment to these different surfaces have not been completely elucidated. At least two types of binding have been described unipolarpolysaccharide-dependent polar attachment and unipolar polysaccharide-independent attachment (both polar and lateral). The genes encoding the enzymes for the production of the former are located on the circular chromosome, while the genes involved in the latter have not been identified. The expression of both of these types of attachment is regulated in response to environmental signals. However, the signals to which they respond differ so that the two types of attachment are not necessarily expressed coordinately. PMID:24926300

  20. An efficient Agrobacterium-mediated transformation system for poplar.

    PubMed

    Movahedi, Ali; Zhang, Jiaxin; Amirian, Rasoul; Zhuge, Qiang

    2014-06-13

    Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone "Nanlin895" (Populus deltoides×P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD600 of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.

  1. The shikonin derivatives and pyrrolizidine alkaloids in hairy root cultures of Lithospermum canescens (Michx.) Lehm.

    PubMed

    Pietrosiuk, A; Sykłowska-Baranek, K; Wiedenfeld, H; Wolinowska, R; Furmanowa, M; Jaroszyk, E

    2006-10-01

    Hairy root cultures of Lithospermum canescens were established using three strains of Agrobacterium rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. Eight lines resulting from infection with A. rhizogenes ATCC 15834 demonstrated sufficient biomass increase and were submitted to further investigations. The contents of acetylshikonin (ACS) and isobutyrylshikonin (IBS) in transformed hairy roots made up ca. 10% of those observed in natural roots of L. canescens (24.35 and 14.48 mg g(-1) DW, respectively). One line, Lc1-D, produced the largest amounts of ACS (2.72 mg g(-1) DW) and IBS (0.307 mg g(-1) DW). Traces of pyrrolizidine alkaloids (PA), canescine and canescenine, were found in all lines of transformed hairy roots.

  2. Bacteriophytochromes control conjugation in Agrobacterium fabrum.

    PubMed

    Bai, Yingnan; Rottwinkel, Gregor; Feng, Juan; Liu, Yiyao; Lamparter, Tilman

    2016-08-01

    Bacterial conjugation, the transfer of single stranded plasmid DNA from donor to recipient cell, is mediated through the type IV secretion system. We performed conjugation assays using a transmissible artificial plasmid as reporter. With this assay, conjugation in Agrobacterium fabrum was modulated by the phytochromes Agp1 and Agp2, photoreceptors that are most sensitive in the red region of visible light. In conjugation studies with wild-type donor cells carrying a pBIN-GUSINT plasmid as reporter that lacked the Ti (tumor inducing) plasmid, no conjugation was observed. When either agp1(-) or agp2(-) knockout donor strains were used, plasmid DNA was delivered to the recipient, indicating that both phytochromes suppress conjugation in the wild type donor. In the recipient strains, the loss of Agp1 or Agp2 led to diminished conjugation. When wild type cells with Ti plasmid and pBIN-GUS reporter plasmid were used as donor, a high rate of conjugation was observed. The DNA transfer was down regulated by red or far-red light by a factor of 3.5. With agp1(-) or agp2(-) knockout donor cells, conjugation in the dark was about 10 times lower than with the wild type donor, and with the double knockout donor no conjugation was observed. These results imply that the phytochrome system has evolved to inhibit conjugation in the light. The decrease of conjugation under different temperature correlated with the decrease of phytochrome autophosphorylation. PMID:27261700

  3. [Agrobacterium-mediated transformation of Cymbidium sinensis].

    PubMed

    Xie, Li; Wang, Fen; Zeng, Ruizhen; Guo, Herong; Zhou, Yuliang; Zhang, Zhisheng

    2015-04-01

    Genetic transformation is an effective method to improve breeding objective traits of orchids. However, there is little information about genetic transformation of Cymbidium sinensis. Rhizomes from shoot-tip culture of C. sinensis cv. 'Qijianbaimo' were used to establish a practical transformation protocol of C. sinensis. Pre-culture time, concentration and treating methods of acetosyringone, concentration of infection bacteria fluid (OD600), infection time, and co-culture time had significant effects on β-glucuronidase (GUS) transient expression rate of C. sinensis cv. 'Qijianbaimo' rhizome. The GUS transient expression rate of rhizome was the highest (11.67%) when rhizomes pre-cultured for 39 d were soaked in bacterium suspension (OD600 = 0.9) supplemented with 200 μmol/L acetosyringone for 35 min, followed by culturing on co-culture medium supplemented with 200 μmol/L acetosyringone for 7 d. Under this transformation conditions, 3 transgenic plantlets, confirmed by GUS histochemical assay and PCR, were obtained from 400 regenerated plantlets, and the genetic transformation rate was 0.75%. This proved that it was feasible to create new cultivars by the use of Agrobacterium-mediated genetic transformation in C. sinense.

  4. Complementary Methodologies To Identify Specific Agrobacterium Strains †

    PubMed Central

    Bouzar, Hacene; Moore, Larry W.

    1987-01-01

    Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field. Images PMID:16347485

  5. Agrobacterium-produced and exogenous cytokinin-modulated Agrobacterium-mediated plant transformation.

    PubMed

    Hwang, Hau-Hsuan; Wang, Ming-Hsuan; Lee, Ying-Ling; Tsai, Yun-Long; Li, Yi-Ho; Yang, Fong-Jhih; Liao, Yu-Chen; Lin, Shao-Kai; Lai, Erh-Min

    2010-09-01

    Agrobacterium tumefaciens is a plant pathogenic bacterium that causes neoplastic growths, called 'crown gall', via the transfer and integration of transferred DNA (T-DNA) from the bacterium into the plant genome. We characterized an acetosyringone (AS)-induced tumour-inducing (Ti) plasmid gene, tzs (trans-zeatin synthesizing), that is responsible for the synthesis of the plant hormone cytokinin in nopaline-type A. tumefaciens strains. The loss of Tzs protein expression and trans-zeatin secretions by the tzs frameshift (tzs-fs) mutant is associated with reduced tumorigenesis efficiency on white radish stems and reduced transformation efficiencies on Arabidopsis roots. Complementation of the tzs-fs mutant with a wild-type tzs gene restored wild-type levels of trans-zeatin secretions and transformation efficiencies. Exogenous application of cytokinin during infection increased the transient transformation efficiency of Arabidopsis roots infected by strains lacking Tzs, which suggests that the lower transformation efficiency resulted from the lack of Agrobacterium-produced cytokinin. Interestingly, although the tzs-fs mutant displayed reduced tumorigenesis efficiency on several tested plants, the loss of Tzs enhanced tumorigenesis efficiencies on green pepper and cowpea. These data strongly suggest that Tzs, by synthesizing trans-zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens. PMID:20696005

  6. Rhizobium pusense is the main human pathogen in the genus Agrobacterium/Rhizobium.

    PubMed

    Aujoulat, F; Marchandin, H; Zorgniotti, I; Masnou, A; Jumas-Bilak, E

    2015-05-01

    Rhizobium pusense was recently described after isolation from the rhizosphere of chickpea. Multilocus sequence-based analysis of clinical isolates identified as Agrobacterium (Rhizobium) radiobacter demonstrated that R. pusense is the main human pathogen within Agrobacterium (Rhizobium) spp. Clinical microbiology of Agrobacterium (Rhizobium) should be considered in the light of recent taxonomic changes.

  7. 77 FR 60431 - Agrobacterium radiobacter strains K84/Kerr-84 and K1026; Notice of Availability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-03

    ... AGENCY Agrobacterium radiobacter strains K84/Kerr-84 and K1026; Notice of Availability AGENCY... final registration review decision for the pesticide Agrobacterium radiobacter strains K84/Kerr-84 and... registration review decision for Agrobacterium radiobacter strains K84/Kerr-84 and K1026, case 4101. When...

  8. Genome Sequence of Ureaplasma diversum Strain ATCC 49782

    PubMed Central

    Marques, Lucas M.; Guimarães, Ana M. S.; Martins, Hellen B.; Rezende, Izadora S.; Barbosa, Maysa S.; Campos, Guilherme B.; do Nascimento, Naíla C.; dos Santos, Andrea P.; Amorim, Aline T.; Santos, Verena M.; Messick, Joanne B.

    2015-01-01

    Here, we report the complete genome sequence of Ureaplasma diversum strain ATCC 49782. This species is of bovine origin, having an association with reproductive disorders in cattle, including placentitis, fetal alveolitis, abortion, and birth of weak calves. It has a small circular chromosome of 975,425 bp. PMID:25883297

  9. Genome Sequence of Ureaplasma diversum Strain ATCC 49782.

    PubMed

    Marques, Lucas M; Guimarães, Ana M S; Martins, Hellen B; Rezende, Izadora S; Barbosa, Maysa S; Campos, Guilherme B; do Nascimento, Naíla C; Dos Santos, Andrea P; Amorim, Aline T; Santos, Verena M; Messick, Joanne B; Timenetsky, Jorge

    2015-04-16

    Here, we report the complete genome sequence of Ureaplasma diversum strain ATCC 49782. This species is of bovine origin, having an association with reproductive disorders in cattle, including placentitis, fetal alveolitis, abortion, and birth of weak calves. It has a small circular chromosome of 975,425 bp.

  10. Draft Genome Sequence of Vibrio (Listonella) anguillarum ATCC 14181

    PubMed Central

    Grim, Christopher J.

    2016-01-01

    We report the draft genome sequence of Vibrio anguillarum ATCC 14181, a Gram-negative, hemolytic, O2 serotype marine bacterium that causes mortality in mariculture species. The availability of this genome sequence will add to our knowledge of diversity and virulence mechanisms of Vibrio anguillarum as well as other pathogenic Vibrio spp.

  11. Draft Genome Sequence of Rhodococcus rhodochrous Strain ATCC 21198

    SciTech Connect

    Shields-Menard, Sara A.; Brown, Steven D; Klingeman, Dawn Marie; Indest, Karl; Hancock, Dawn; Wewalwela, Jayani; French, Todd; Donaldson, Janet

    2014-01-01

    Rhodococcus rhodochrous is a Gram-positive red-pigmented bacterium commonly found in the soil. The draft genome sequence for R. rhodochrous strain ATCC 21198 is presented here to provide genetic data for a better understanding of its lipid-accumulating capabilities.

  12. Draft Genome Sequence of Mycobacterium interjectum Strain ATCC 51457T

    PubMed Central

    Levasseur, Anthony; Asmar, Shady; Robert, Catherine

    2016-01-01

    Mycobacterium interjectum is a nontuberculosis species rarely responsible for human infection. The draft genome of M. interjectum ATCC 51457T comprises 5,927,979 bp, exhibiting 67.91% G+C content, 5,314 protein-coding genes, and 51 predicted RNA genes. PMID:27231376

  13. Draft Genome Sequence of Mycobacterium houstonense Strain ATCC 49403T

    PubMed Central

    Levasseur, Anthony; Asmar, Shady; Robert, Catherine

    2016-01-01

    Mycobacterium houstonense is a nontuberculous species rarely responsible for human infection. The draft genome of M. houstonense ATCC 49403T comprises 6,451,020 bp, exhibiting a 66.96% G+C content, 5,881 protein-coding genes, and 65 predicted RNA genes. PMID:27231371

  14. Draft Genome Sequence of Bacillus megaterium Type Strain ATCC 14581

    PubMed Central

    Arya, Gitanjali; Petronella, Nicholas; Crosthwait, Jennifer; Carrillo, Catherine D.

    2014-01-01

    Bacillus megaterium is a Gram-positive, rod-shaped, spore-forming bacterium of biotechnological importance. Here, we report a 5.7-Mbp draft genome sequence of B. megaterium ATCC 14581, which is the type strain of the species. PMID:25395629

  15. Draft Genome Sequence of Bacillus megaterium Type Strain ATCC 14581.

    PubMed

    Arya, Gitanjali; Petronella, Nicholas; Crosthwait, Jennifer; Carrillo, Catherine D; Shwed, Philip S

    2014-01-01

    Bacillus megaterium is a Gram-positive, rod-shaped, spore-forming bacterium of biotechnological importance. Here, we report a 5.7-Mbp draft genome sequence of B. megaterium ATCC 14581, which is the type strain of the species. PMID:25395629

  16. Complete genome sequence of Campylobacter gracilis ATCC 33236T

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The human oral pathogen Campylobacter gracilis has been isolated from periodontal and endodontal infections, and also from non-oral head, neck or lung infections. This study describes the whole-genome sequence of the human periodontal isolate ATCC 33236T (=FDC 1084), which is the first closed genome...

  17. Draft Genome Sequence of Alicyclobacillus acidoterrestris Strain ATCC 49025

    PubMed Central

    Pasvolsky, Ronit; Sela, Noa; Green, Stefan J.; Zakin, Varda

    2013-01-01

    Alicyclobacillus acidoterrestris is a spore-forming Gram-positive, thermo-acidophilic, nonpathogenic bacterium which contaminates commercial pasteurized fruit juices. The draft genome sequence for A. acidoterrestris strain ATCC 49025 is reported here, providing genetic data relevant to the successful adaptation and survival of this strain in its ecological niche. PMID:24009113

  18. Multilocus sequence-based analysis delineates a clonal population of Agrobacterium (Rhizobium) radiobacter (Agrobacterium tumefaciens) of human origin.

    PubMed

    Aujoulat, Fabien; Jumas-Bilak, Estelle; Masnou, Agnès; Sallé, Fanny; Faure, Denis; Segonds, Christine; Marchandin, Hélène; Teyssier, Corinne

    2011-05-01

    The genus Agrobacterium includes plant-associated bacteria and opportunistic human pathogens. Taxonomy and nomenclature within the genus remain controversial. In particular, isolates of human origin were all affiliated with the species Agrobacterium (Rhizobium) radiobacter, while phytopathogenic strains were designated under the synonym denomination Agrobacterium tumefaciens. In order to study the relative distribution of Agrobacterium strains according to their origins, we performed a multilocus sequence-based analysis (MLSA) on a large collection of 89 clinical and environmental strains from various origins. We proposed an MLSA scheme based on the partial sequence of 7 housekeeping genes (atpD, zwf, trpE, groEL, dnaK, glnA, and rpoB) present on the circular chromosome of A. tumefaciens C58. Multilocus phylogeny revealed that 88% of the clinical strains belong to genovar A7, which formed a homogeneous population with linkage disequilibrium, suggesting a low rate of recombination. Comparison of genomic fingerprints obtained by pulsed-field gel electrophoresis (PFGE) showed that the strains of genovar A7 were epidemiologically unrelated. We present genetic evidence that genovar A7 may constitute a human-associated population distinct from the environmental population. Also, phenotypic characteristics, such as culture at 42°C, agree with this statement. This human-associated population might represent a potential novel species in the genus Agrobacterium.

  19. Draft Genome Sequence of Strain ATCC 33958, Reported To Be Elizabethkingia miricola

    PubMed Central

    Matyi, Stephanie A.; Hoyt, Peter R.; Ayoubi-Canaan, Patricia; Hasan, Nabeeh A.

    2015-01-01

    We report the draft genome of Elizabethkingia strain ATCC 33958, which has been classified as Elizabethkingia miricola. Similar to other Elizabethkingia species, the ATCC 33958 draft genome contains numerous β-lactamase genes. ATCC 33958 also harbors a urease gene cluster which supports classification as E. miricola. PMID:26205869

  20. 40 CFR 180.1205 - Beauveria bassiana ATCC #74040; exemption from the requirements of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Beauveria bassiana ATCC #74040... RESIDUES IN FOOD Exemptions From Tolerances § 180.1205 Beauveria bassiana ATCC #74040; exemption from the... the insecticide Beauveria bassiana (ATCC #74040) in or on all food commodities when applied or used...

  1. 40 CFR 180.1205 - Beauveria bassiana ATCC #74040; exemption from the requirements of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Beauveria bassiana ATCC #74040... RESIDUES IN FOOD Exemptions From Tolerances § 180.1205 Beauveria bassiana ATCC #74040; exemption from the... the insecticide Beauveria bassiana (ATCC #74040) in or on all food commodities when applied or used...

  2. Vitreoscilla hemoglobin promotes Salecan production by Agrobacterium sp. ZX09*

    PubMed Central

    Chen, Yun-mei; Xu, Hai-yang; Wang, Yang; Zhang, Jian-fa; Wang, Shi-ming

    2014-01-01

    Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. ZX09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan a promising material for applications in coagulation, lubrication, protection against acute liver injury, and alleviating constipation. In this study, we cloned the Vitreoscilla hemoglobin gene into a broad-host-range plasmid pCM158. Without antibiotic selection, there was negligible loss of the plasmid in the host Agrobacterium sp. ZX09 after one passage of cultivation. The expression of Vitreoscilla hemoglobin was demonstrated by carbon monoxide (CO) difference spectrum. The engineered strain Agrobacterium sp. ZX09 increased Salecan yield by 30%. The other physiological changes included its elevated respiration rate and cellular invertase activity. PMID:25367790

  3. In planta Agrobacterium-mediated transformation by vacuum infiltration.

    PubMed

    Tague, Brian W; Mantis, Joanna

    2006-01-01

    In planta Agrobacterium-mediated transformation using vacuum infiltration results in transgenic Arabidopsis thaliana without the use of sterile conditions or plant regeneration. Plants are grown in pots, in standard potting mix. Agrobacterium tumefaciens, carrying an appropriate plant transformation vector, is suspended in an infiltration medium that contains, at a minimum, sucrose and the surfactant Silwet L-77. Flower buds are immersed in the suspension of A. tumefaciens. The application of a vacuum drives the bacteria into the intercellular air spaces. A portion of the Agrobacterium Ti plasmid known as the T-DNA region, which has been engineered to carry a selectable marker, becomes integrated into the plant genomic DNA. Plants are allowed to set seed. Seeds are germinated in selective conditions to recover transformants. PMID:16739579

  4. Spectrophotometric evaluation of selenium binding by Saccharomyces cerevisiae ATCC MYA-2200 and Candida utilis ATCC 9950 yeast.

    PubMed

    Kieliszek, Marek; Błażejak, Stanisław; Płaczek, Maciej

    2016-05-01

    In this study, the ability of selenium binding the biomas of Saccharomyces cerevisiae ATCC MYA-2200 and Candida utilis ATCC 9950 was investigated. Sodium selenite(IV) salts were added to the experimental media at concentrations of 10, 20, 40, and 60 mg Se(4+) L(-1). In the tested concentration range, one concentration reported a significant reduction in the biomass yield of both yeast strains. Intense growth was observed for C. utilis yeast, which reached the highest biomass yield of 15 gd.w.L(-1) after 24h cultivation in the presence of 10mg Se(4+) L(-1). Based on the use of spectrophotometric method for the determination of selenium content by using Variamine Blue as a chromogenic agent, efficient accumulation of this element in the biomass of the investigated yeast was observed. The highest amount of selenium, that is, 5.64 mg Se(4+)gd.w.(-1), was bound from the environment by S. cerevisiae ATCC MYA-2200 cultured in the presence of 60 mg Se(4+) L(-1) medium 72h Slightly less amount, 5.47 mg Se(4+) gd.w.(-1), was absorbed by C. utilis ATCC 9950 during similar cultural conditions. Based on the results of the biomass yield and the use of selenium from the medium, it can be observed that yeasts of the genus Candida are more efficient in binding this element, and this property finds practical application in the production of selenium-enriched yeast.

  5. Spectrophotometric evaluation of selenium binding by Saccharomyces cerevisiae ATCC MYA-2200 and Candida utilis ATCC 9950 yeast.

    PubMed

    Kieliszek, Marek; Błażejak, Stanisław; Płaczek, Maciej

    2016-05-01

    In this study, the ability of selenium binding the biomas of Saccharomyces cerevisiae ATCC MYA-2200 and Candida utilis ATCC 9950 was investigated. Sodium selenite(IV) salts were added to the experimental media at concentrations of 10, 20, 40, and 60 mg Se(4+) L(-1). In the tested concentration range, one concentration reported a significant reduction in the biomass yield of both yeast strains. Intense growth was observed for C. utilis yeast, which reached the highest biomass yield of 15 gd.w.L(-1) after 24h cultivation in the presence of 10mg Se(4+) L(-1). Based on the use of spectrophotometric method for the determination of selenium content by using Variamine Blue as a chromogenic agent, efficient accumulation of this element in the biomass of the investigated yeast was observed. The highest amount of selenium, that is, 5.64 mg Se(4+)gd.w.(-1), was bound from the environment by S. cerevisiae ATCC MYA-2200 cultured in the presence of 60 mg Se(4+) L(-1) medium 72h Slightly less amount, 5.47 mg Se(4+) gd.w.(-1), was absorbed by C. utilis ATCC 9950 during similar cultural conditions. Based on the results of the biomass yield and the use of selenium from the medium, it can be observed that yeasts of the genus Candida are more efficient in binding this element, and this property finds practical application in the production of selenium-enriched yeast. PMID:27049131

  6. Two-way chemical signaling in Agrobacterium-plant interactions.

    PubMed Central

    Winans, S C

    1992-01-01

    The discovery in 1977 that Agrobacterium species can transfer a discrete segment of oncogenic DNA (T-DNA) to the genome of host plant cells has stimulated an intense interest in the molecular biology underlying these plant-microbe associations. This attention in turn has resulted in a series of insights about the biology of these organisms that continue to accumulate at an ever-increasing rate. This excitement was due in part to the notion that this unprecedented interkingdom DNA transfer could be exploited to create transgenic plants containing foreign genes of scientific or commercial importance. In the course of these discoveries, Agrobacterium became one of the best available models for studying the molecular interactions between bacteria and higher organisms. One extensively studied aspect of this association concerns the exchange of chemical signals between Agrobacterium spp. and host plants. Agrobacterium spp. can recognize no fewer than five classes of low-molecular-weight compounds released from plants, and other classes probably await discovery. The most widely studied of these are phenolic compounds, which stimulate the transcription of the genes needed for infection. Other compounds include specific monosaccharides and acidic environments which potentiate vir gene induction, acidic polysaccharides which induce one or more chromosomal genes, and a family of compounds called opines which are released from tumorous plant cells to the bacteria as nutrient sources. Agrobacterium spp. in return release a variety of chemical compounds to plants. The best understood is the transferred DNA itself, which contains genes that in various ways upset the balance of phytohormones, ultimately causing neoplastic cell proliferation. In addition to transferring DNA, some Agrobacterium strains directly secrete phytohormones. Finally, at least some strains release a pectinase, which degrades a component of plant cell walls. PMID:1579105

  7. Biodegradation of crystal violet by Agrobacterium radiobacter.

    PubMed

    Parshetti, G K; Parshetti, S G; Telke, A A; Kalyani, D C; Doong, R A; Govindwar, S P

    2011-01-01

    Agrobacterium radiobacter MTCC 8161 completely decolorized the Crystal Violet with 8 hr (10 mg/L) at static anoxic conditions. The decreased decolorization capability by A. radiobacter was observed, when the Crystal Violet concentration was increased from 10 to 100 mg/L. Semi-synthetic medium containing 1% yeast extract and 0.1% NH4C1 has shown 100% decolorization of Crystal Violet within 5 hr. A complete degradation of Crystal Violet by A. radiobacter was observed up to 7 cycles of repeated addition (10 mg/L). When the effect of increasing inoculum concentration on decolorization of Crystal Violet (100 mg/L) was studied, maximum decolorization was observed with 15% inoculum concentration. A significant increase in the activities of laccase (184%) and aminopyrine N-demethylase (300%) in cells obtained after decolorization indicated the involvement of these enzymes in decolorization process. The intermediates formed during the degradation of Crystal Violet were analyzed by gas chromatography and mass spectroscopy (GC/MS). It was detected the presence of N,N,N',N"-tetramethylpararosaniline, [N, N-dimethylaminophenyl] [N-methylaminophenyl] benzophenone, N, N-dimethylaminobenzaldehyde, 4-methyl amino phenol and phenol. We proposed the hypothetical metabolic pathway of Crystal Violet biodegradation by A. radiobacter. Phytotoxicity and microbial toxicity study showed that Crystal Violet biodegradation metabolites were less toxic to bacteria (A. radiobacter, P. aurugenosa and A. vinelandii) contributing to soil fertility and for four kinds of plants (Sorghum bicolor Vigna radiata, Lens culinaris and Triticum aestivum) which are most sensitive, fast growing and commonly used in Indian agriculture.

  8. Complete Genome Sequence of Agrobacterium tumefaciens Ach5.

    PubMed

    Huang, Ya-Yi; Cho, Shu-Ting; Lo, Wen-Sui; Wang, Yi-Chieh; Lai, Erh-Min; Kuo, Chih-Horng

    2015-01-01

    Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease. The strain Ach5 was isolated from yarrow (Achillea ptarmica L.) and is the wild-type progenitor of other derived strains widely used for plant transformation. Here, we report the complete genome sequence of this bacterium. PMID:26044425

  9. Trojan horse strategy in Agrobacterium transformation: abusing MAPK defense signaling.

    PubMed

    Djamei, Armin; Pitzschke, Andrea; Nakagami, Hirofumi; Rajh, Iva; Hirt, Heribert

    2007-10-19

    Nuclear import of transfer DNA (T-DNA) is a central event in Agrobacterium transformation of plant cells and is thought to occur by the hijacking of certain host cell proteins. The T-DNA-associated virulence protein VirE2 mediates this process by binding to the nuclear import machinery via the host cell factor VIP1, whose role in plants has been so far unknown. Here we show that VIP1 is a transcription factor that is a direct target of the Agrobacterium-induced mitogen-activated protein kinase (MAPK) MPK3. Upon phosphorylation by MPK3, VIP1 relocalizes from the cytoplasm to the nucleus and regulates the expression of the PR1 pathogenesis-related gene. MAPK-dependent phosphorylation of VIP1 is necessary for VIP1-mediated Agrobacterium T-DNA transfer, indicating that Agrobacterium abuses the MAPK-targeted VIP1 defense signaling pathway for nuclear delivery of the T-DNA complex as a Trojan horse. PMID:17947581

  10. Impact of biological amendments on Agrobacterium tumefaciens soil survival

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paradox, the primary walnut rootstock used in California, is susceptible to Agrobacterium tumefaciens, which causes crown gall. While A. tumefaciens is susceptible to commonly used fumigants such as methyl bromide (MeBr) and Telone-C35 (1,3-dichloropropene and chloropicrin), these fumigants also sig...

  11. Virulence of Agrobacterium tumefaciens strain A281 on legumes

    SciTech Connect

    Hood, E.E.; Fraley, R.T.; Chilton, M.D.

    1987-03-01

    This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an L,L-succinamopine strain. The authors tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains.

  12. [Agrobacterium rubi strains from blueberry plants are highly diverse].

    PubMed

    Abrahamovich, Eliana; López, Ana C; Alippi, Adriana M

    2014-01-01

    The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplified 23S rDNA showed high phenotypic and genotypic intraspecific variation.

  13. Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.

    PubMed

    Sparks, Caroline A; Doherty, Angela; Jones, Huw D

    2014-01-01

    The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.). PMID:24243208

  14. Is VIP1 important for Agrobacterium-mediated transformation?

    PubMed

    Shi, Yong; Lee, Lan-Ying; Gelvin, Stanton B

    2014-09-01

    Agrobacterium genetically transforms plants by transferring and integrating T-(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus-tagged VirE2 or Venus-tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1-Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY-2 protoplasts, regardless of whether VirE2 was co-expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium-mediated transformation or VirE2 subcellular localization.

  15. Agrobacterium-mediated genetic transformation of Prunus salicina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report Agrobacterium tumefaciens-mediated transformation from hypocotyls slices of two Prunus salicina varieties, 'Angeleno' and 'Larry Anne', using a modification of the technique previously described for P. domestica. Regeneration rates on thidiazuron (TDZ) and indole-3-butyric acid (IBA) supp...

  16. Draft Genome Sequence of Mycobacterium chelonae Type Strain ATCC 35752

    PubMed Central

    Hasan, Nabeeh A.; Davidson, Rebecca M.; de Moura, Vinicius Calado Nogueira; Garcia, Benjamin J.; Reynolds, Paul R.; Epperson, L. Elaine; Farias-Hesson, Eveline; DeGroote, Mary Ann; Jackson, Mary

    2015-01-01

    Mycobacterium chelonae is a rapidly growing opportunistic nontuberculous mycobacterial (NTM) species that causes infections in humans and other hosts. Here, we report the draft genome sequence of Mycobacterium chelonae type strain ATCC 35752, consisting of 4.89 Mbp, 63.96% G+C content, 4,489 protein-coding genes, 48 tRNAs, and 3 rRNA genes. PMID:26021923

  17. Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC

    DOEpatents

    Adney, W.S.; Thomas, S.R.; Nieves, R.A.; Himmel, M.E.

    1994-11-22

    A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C[sub 1], and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81 C at pH's from about 2 to about 9, and at a inactivation temperature of about 100 C at pH's from about 2 to about 9. 9 figs.

  18. Magnetic response in cultures of Streptococcus mutans ATCC-27607.

    PubMed

    Adamkiewicz, V W; Bassous, C; Morency, D; Lorrain, P; Lepage, J L

    1987-01-01

    Streptococcus mutans ATCC-27607 produces exopolysaccharides that adhere to glass. In the normal geomagnetic field about 50% more polysaccharide adhere preferentially to glass surfaces facing North as compared to South facing surfaces. Reversal of the direction of the magnetic field by 180 degrees produces a similar reversal in the direction of the preferential accumulation. Reduction of the field by 90% abolishes the preferential accumulation. PMID:3582582

  19. Temperature Effects on Agrobacterium Phytochrome Agp1

    PubMed Central

    Njimona, Ibrahim; Lamparter, Tilman

    2011-01-01

    Phytochromes are widely distributed biliprotein photoreceptors with a conserved N-terminal chromophore-binding domain. Most phytochromes bear a light-regulated C-terminal His kinase or His kinase-like region. We investigated the effects of light and temperature on the His kinase activity of the phytochrome Agp1 from Agrobacterium tumefaciens. As in earlier studies, the phosphorylation activity of the holoprotein after far-red irradiation (where the red-light absorbing Pr form dominates) was stronger than that of the holoprotein after red irradiation (where the far red-absorbing Pfr form dominates). Phosphorylation activities of the apoprotein, far red-irradiated holoprotein, and red-irradiated holoprotein decreased when the temperature increased from 25°C to 35°C; at 40°C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40°C was nearly completely restored when the temperature returned to 25°C. UV/visible spectroscopy indicated that the protein was not denatured up to 45°C. At 50°C, however, Pfr denatured faster than the dark-adapted sample containing the Pr form of Agp1. The Pr visible spectrum was unaffected by temperatures of 20–45°C, whereas irradiated samples exhibited a clear temperature effect in the 30–40°C range in which prolonged irradiation resulted in the photoconversion of Pfr into a new spectral species termed Prx. Pfr to Prx photoconversion was dependent on the His-kinase module of Agp1; normal photoconversion occurred at 40°C in the mutant Agp1-M15, which lacks the C-terminal His-kinase module, and in a domain-swap mutant in which the His-kinase module of Agp1 is replaced by the His-kinase/response regulator module of the other A. tumefaciens phytochrome, Agp2. The temperature-dependent kinase activity and spectral properties in the physiological temperature range suggest that Agp1 serves as an integrated light and temperature sensor in A. tumefaciens. PMID:22043299

  20. Plant defense pathways subverted by Agrobacterium for genetic transformation

    PubMed Central

    Krichevsky, Alexander; Kozlovsky, Stanislav V; Yasmin, Farzana; Citovsky, Vitaly

    2010-01-01

    The soil phytopathogen Agrobacterium has the unique ability to introduce single-stranded transferred DNA (T-DNA) from its tumor-inducing (Ti) plasmid into the host cell in a process known as horizontal gene transfer. Following its entry into the host cell cytoplasm, the T-DNA associates with the bacterial virulence (Vir) E2 protein, also exported from Agrobacterium, creating the T-DNA nucleoprotein complex (T-complex), which is then translocated into the nucleus where the DNA is integrated into the host chromatin. VirE2 protects the T-DNA from the host DNase activities, packages it into a helical filament and interacts with the host proteins, one of which, VIP1, facilitates nuclear import of the T-complex and its subsequent targeting to the host chromatin. Although the VirE2 and VIP1 protein components of the T-complex are vital for its intracellular transport, they must be removed to expose the T-DNA for integration. Our recent work demonstrated that this task is aided by an host defense-related F-box protein VBF that is induced by Agrobacterium infection and that recognizes and binds VIP1. VBF destabilizes VirE2 and VIP1 in yeast and plant cells, presumably via SCF-mediated proteasomal degradation. VBF expression in and export from the Agrobacterium cell lead to increased tumorigenesis. Here, we discuss these findings in the context of the “arms race” between Agrobacterium infectivity and plant defense. PMID:20890133

  1. Cellulose produced by Gluconacetobacter xylinus strains ATCC 53524 and ATCC 23768: Pellicle formation, post-synthesis aggregation and fiber density.

    PubMed

    Lee, Christopher M; Gu, Jin; Kafle, Kabindra; Catchmark, Jeffrey; Kim, Seong H

    2015-11-20

    The pellicle formation, crystallinity, and bundling of cellulose microfibrils produced by bacterium Gluconacetobacter xylinus were studied. Cellulose pellicles were produced by two strains (ATCC 53524 and ATCC 23769) for 1 and 7 days; pellicles were analyzed with scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrational sum-frequency-generation (SFG) spectroscopy, and attenuated total reflectance infrared (ATR-IR) spectroscopy. The bacterial cell population was higher at the surface exposed to air, indicating that the newly synthesized cellulose is deposited at the top of the pellicle. XRD, ATR-IR, and SFG analyses found no significant changes in the cellulose crystallinity, crystal size or polymorphic distribution with the culture time. However, SEM and SFG analyses revealed cellulose macrofibrils produced for 7 days had a higher packing density at the top of the pellicle, compared to the bottom. These findings suggest that the physical properties of cellulose microfibrils are different locally within the bacterial pellicles. PMID:26344281

  2. Cellulose produced by Gluconacetobacter xylinus strains ATCC 53524 and ATCC 23768: Pellicle formation, post-synthesis aggregation and fiber density.

    PubMed

    Lee, Christopher M; Gu, Jin; Kafle, Kabindra; Catchmark, Jeffrey; Kim, Seong H

    2015-11-20

    The pellicle formation, crystallinity, and bundling of cellulose microfibrils produced by bacterium Gluconacetobacter xylinus were studied. Cellulose pellicles were produced by two strains (ATCC 53524 and ATCC 23769) for 1 and 7 days; pellicles were analyzed with scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrational sum-frequency-generation (SFG) spectroscopy, and attenuated total reflectance infrared (ATR-IR) spectroscopy. The bacterial cell population was higher at the surface exposed to air, indicating that the newly synthesized cellulose is deposited at the top of the pellicle. XRD, ATR-IR, and SFG analyses found no significant changes in the cellulose crystallinity, crystal size or polymorphic distribution with the culture time. However, SEM and SFG analyses revealed cellulose macrofibrils produced for 7 days had a higher packing density at the top of the pellicle, compared to the bottom. These findings suggest that the physical properties of cellulose microfibrils are different locally within the bacterial pellicles.

  3. Reconciliation of Sequence Data and Updated Annotation of the Genome of Agrobacterium tumefaciens C58, and Distribution of a Linear Chromosome in the Genus Agrobacterium

    PubMed Central

    Slater, Steven; Setubal, João C.; Houmiel, Kathryn; Sun, Jian; Kaul, Rajinder; Goldman, Barry S.; Farrand, Stephen K.; Almeida, Nalvo; Burr, Thomas; Nester, Eugene; Rhoads, David M.; Kadoi, Ryosuke; Ostheimer, Trucian; Pride, Nicole; Sabo, Allison; Henry, Erin; Telepak, Erin; Cromes, Lindsey; Harkleroad, Alana; Oliphant, Louis; Pratt-Szegila, Phil; Welch, Roy; Wood, Derek

    2013-01-01

    Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium. PMID:23241979

  4. Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium-plant interaction.

    PubMed

    Xu, X Q; Li, L P; Pan, S Q

    2001-11-01

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

  5. [Use of highly dispersed materials for culturing and isolation of granular Agrobacterium radiobacter preparations ].

    PubMed

    Kurdish, I K; Titova, L V

    2001-01-01

    The effects of synthetic and natural high-dispersion materials on the growth of Agrobacterium radiobacter were studied. Natural minerals montmorillonite and palygorskite (10 g/l nutrient medium) were more potent than high-dispersion silica and its modified forms in stimulating growth of Agrobacterium radiobacter. The interaction of Agrobacterium radiobacter with clay minerals increased the survival rate of bacteria at supraoptimal temperatures. We elaborated new granular bacterial preparation, which enhanced the productivity of cucumbers by 12-15%.

  6. Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068

    DOEpatents

    Himmel, Michael E.; Adney, William S.; Tucker, Melvin P.; Grohmann, Karel

    1994-01-01

    A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068). The cellulase is water soluble, possesses both C.sub.1 and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83.degree. C. at pH's from about 2 to about 9, and in inactivation temperature of about 110.degree. C. at pH's from about 2 to about 9.

  7. Draft genome sequence of Rhodococcus rhodochrous strain ATCC 17895

    PubMed Central

    Chen, Bi-Shuang; Otten, Linda G.; Resch, Verena; Muyzer, Gerard; Hanefeld, Ulf

    2013-01-01

    Rhodococcus rhodochrous ATCC 17895 possesses an array of mono- and dioxygenases, as well as hydratases, which makes it an interesting organism for biocatalysis. R. rhodochrous is a Gram-positive aerobic bacterium with a rod-like morphology. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Based on small subunit rRNA analysis, the strain is more likely to be a strain of Rhodococcus erythropolis rather than Rhodococcus rhodochrous. PMID:24501654

  8. Thermostable purified endoglucanase from Acidothermus cellulolyticus ATCC 43068

    DOEpatents

    Himmel, M.E.; Adney, W.S.; Tucker, M.P.; Grohmann, K.

    1994-01-04

    A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068) is presented. The cellulase is water soluble, possesses both C[sub 1] and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83 C at pH's from about 2 to about 9, and in inactivation temperature of about 110 C at pH's from about 2 to about 9. 7 figures.

  9. Agrobacterium-mediated genetic transformation of Prunus salicina.

    PubMed

    Urtubia, Carolina; Devia, Jessica; Castro, Alvaro; Zamora, Pablo; Aguirre, Carlos; Tapia, Eduardo; Barba, Paola; Dell Orto, Paola; Moynihan, Michael R; Petri, César; Scorza, Ralph; Prieto, Humberto

    2008-08-01

    We report Agrobacterium tumefaciens-mediated transformation of two Prunus salicina varieties, 'Angeleno' and 'Larry Anne', using a modification of the hypocotyl slice technique previously described for P. domestica. Regeneration rates on thidiazuron (TDZ) and indole-3-butyric acid (IBA) supplemented Murashige and Skoog (MS) media reached 11% for 'Angeleno' and 19% for 'Larry Anne' hypocotyl slices. Transformation using Agrobacterium tumefaciens GV3101 harboring a plasmid with the neomycin phosphotransferase II (nptII) and the green fluorescent protein (gfp) genes produced ten independent lines, six from 'Angeleno' and four from 'Larry Anne', representing transformation efficiencies of 0.8 and 0.3%, respectively, relative to the initial number of hypocotyl slices. Plants of six lines were found to produce the transgene encoded mRNAs. DNA blotting demonstrated the presence of transgene sequences in trees from five lines after 18 months of growth in the greenhouse.

  10. Agrobacterium-mediated transformation of maize (Zea mays) immature embryos.

    PubMed

    Lee, Hyeyoung; Zhang, Zhanyuan J

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is one of the most efficient and simple gene delivery systems for genetic improvement and biology studies in maize. This system has become more widely used by both public and private laboratories. However, transformation efficiencies vary greatly from laboratory to laboratory for the same genotype. Here, we illustrate our advanced Agrobacterium-mediated transformation method in Hi-II maize using simple binary vectors. The protocol utilizes immature embryos as starting explants and the bar gene as a selectable marker coupled with bialaphos as a selective agent. The protocol offers efficient transformation results with high reproducibility, provided that some experimental conditions are well controlled. This transformation method, with minor modifications, can be also employed to transform certain maize inbreds.

  11. Agrobacterium-mediated transformation of three freshwater microalgal strains.

    PubMed

    Sanitha, Mary; Radha, Sudhakar; Fatima, Anwar Aliya; Devi, Selvaraju Gayathri; Ramya, Mohandass

    2014-01-01

    Microalgal transformation has gained interest in recent years. Agrobacterium-mediated transformation remains as the most efficient method for the development of transgenic plants and microalgae due to its wide host range, inexpensive procedure and transfer of large segments of DNA. In the present study, three different microalgal species were isolated from freshwater environment and identified based on the morphological characteristics and ITS-2 region amplification. Agrobacterium-mediated transformation was successful for the isolates Chlorella sp., Ankistrodesmus sp and Scenedesmus bajacalifornicus. Gene integration and expression was confirmed by PCR amplification of hptII and GUS histochemical assay. A. tumifaciens contamination was checked by amplification of npt II gene (kanamycin resistant) which lies outside the T-border. Based on GUS assay, transformation efficiencies were found to be 12.25% for Chlorella sp. 2.96% for Scenedesmus bajacalifornicus and 3.5% for Ankistrodesmus sp.

  12. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  13. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  14. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-05-26

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  15. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  16. Agrobacterium-mediated transformation of barley (Hordeum vulgare L.).

    PubMed

    Ismagul, Ainur; Mazonka, Iryna; Callegari, Corinne; Eliby, Serik

    2014-01-01

    Barley biotechnology requires efficient genetic engineering tools for producing transgenic plants necessary for conducting reverse genetics analyses in breeding and functional genomics research. Agrobacterium-mediated genetic transformation is an important technique for producing barley transgenics with simple low-copy number transgenes. This chapter reports a refined protocol for the systematic high-throughput transformation of the advanced Australian spring barley breeding line WI4330.

  17. Plant responses to Agrobacterium tumefaciens and crown gall development.

    PubMed

    Gohlke, Jochen; Deeken, Rosalia

    2014-01-01

    Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumors. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide ("omic") approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumor formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic, and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant. PMID:24795740

  18. Characterization of Agrobacterium tumefaciens DNA ligases C and D.

    PubMed

    Zhu, Hui; Shuman, Stewart

    2007-01-01

    Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3'-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3'-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3'-OH end. The PE domains also have a 3'-phosphatase activity on an all-DNA primer-template that yields a 3'-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ.

  19. Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea.

    PubMed

    Bhalla, Prem L; Singh, Mohan B

    2008-01-01

    Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe an efficient protocol for Agrobacterium-mediated transformation using seedling explants that is applicable to various Brassica varieties; this protocol has been used to genetically engineer commercial cultivars of canola and cauliflower in our laboratory. Young seedling explants are inoculated with Agrobacterium on the day of explant preparation. Explants are grown for 1 week in the absence of a selective agent before being transferred to a selective medium to recover transgenic shoots. Transgenic shoots are subjected to an additional round of selection on medium containing higher levels of the selective agent and a low-carbohydrate source; this helps to eliminate false-positive plants. Use of seedling explants offers flexible experiment planning and a convenient explant source. Using this protocol, transgenic plants can be obtained in 2.5 to 3.5 months.

  20. Production of Biohydrogen from Wastewater by Klebsiella oxytoca ATCC 13182.

    PubMed

    Thakur, Veena; Tiwari, K L; Jadhav, S K

    2015-08-01

    Production of biohydrogen from distillery effluent was carried out by using Klebsiella oxytoca ATCC 13182. The work focuses on optimization of pH, temperature, and state of bacteria, which are the various affecting factors for fermentative biohydrogen production. Results indicates that at 35 °C for suspended cultures, the production was at its maximum (i.e., 91.33 ± 0.88 mL) when compared with other temperatures. At 35 °C and at pH 5 and 6, maximum productions of 117.67 ± 1.45 and 111.67 ± 2.72 mL were observed with no significant difference. When immobilized, Klebsiella oxytoca ATCC 13182 was used for biohydrogen production at optimized conditions, production was 186.33 ± 3.17 mL. Hence, immobilized cells were found to be more advantageous for biological hydrogen production over suspended form. Physicochemical analysis of the effluent was conducted before and after fermentation and the values suggested that the fermentative process is an efficient method for biological treatment of wastewater. PMID:26237683

  1. Production of Biohydrogen from Wastewater by Klebsiella oxytoca ATCC 13182.

    PubMed

    Thakur, Veena; Tiwari, K L; Jadhav, S K

    2015-08-01

    Production of biohydrogen from distillery effluent was carried out by using Klebsiella oxytoca ATCC 13182. The work focuses on optimization of pH, temperature, and state of bacteria, which are the various affecting factors for fermentative biohydrogen production. Results indicates that at 35 °C for suspended cultures, the production was at its maximum (i.e., 91.33 ± 0.88 mL) when compared with other temperatures. At 35 °C and at pH 5 and 6, maximum productions of 117.67 ± 1.45 and 111.67 ± 2.72 mL were observed with no significant difference. When immobilized, Klebsiella oxytoca ATCC 13182 was used for biohydrogen production at optimized conditions, production was 186.33 ± 3.17 mL. Hence, immobilized cells were found to be more advantageous for biological hydrogen production over suspended form. Physicochemical analysis of the effluent was conducted before and after fermentation and the values suggested that the fermentative process is an efficient method for biological treatment of wastewater.

  2. Agrobacterium-mediated transformation of Mexican lime (Citrus aurantifolia Swingle) using optimized systems for epicotyls and cotelydons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epicotyl and internodal stem segments provide the predominantly used explants for regeneration of transgenic citrus plants following co-cultivation with Agrobacterium. Previous reports using epicotyls segments from Mexican lime have shown low affinity for Agrobacterium tumefaciens infection which re...

  3. Antibacterial Activity of Synthetic Peptides Derived from Lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212

    PubMed Central

    León-Calvijo, María A.; Leal-Castro, Aura L.; Almanzar-Reina, Giovanni A.; Rosas-Pérez, Jaiver E.; García-Castañeda, Javier E.; Rivera-Monroy, Zuly J.

    2015-01-01

    Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4–33 μM) and E. faecalis (MIC 10–33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield. PMID:25815317

  4. Pseudomonas oleovorans subsp. lubricantis subsp. nov., and reclassification of Pseudomonas pseudoalcaligenes ATCC 17440T as later synonym of Pseudomonas oleovorans ATCC 8062 T.

    PubMed

    Saha, Ratul; Spröer, Cathrin; Beck, Brian; Bagley, Susan

    2010-04-01

    Isolate RS1(T) isolated from used metalworking fluid was found to be a Gram-negative, motile, and non-spore forming rod. Based on phylogenetic analyses with 16S rRNA, isolate RS1(T) was placed into the mendocina sublineage of Pseudomonas. The major whole cell fatty acids were C(18:1)omega7c (32.6%), C(16:0) (25.5%), and C(15:0) ISO 2OH/C(16:1)omega7c (14.4%). The sequence similarities of isolate RS1(T) based on gyrB and rpoD genes were 98.9 and 98.0% with Pseudomonas pseudoalcaligenes, and 98.5 and 98.1% with Pseudomonas oleovorans, respectively. The ribotyping pattern showed a 0.60 similarity with P. oleovorans ATCC 8062(T) and 0.63 with P. pseudoalcaligenes ATCC17440(T). The DNA G + C content of isolate RS1(T) was 62.2 mol.%. The DNA-DNA relatedness was 73.0% with P. oleovorans ATCC 8062(T) and 79.1% with P. pseudoalcaligenes ATCC 17440(T). On the basis of morphological, biochemical, and molecular studies, isolate RS1(T) is considered to represent a new subspecies of P. oleovorans. Furthermore, based on the DNA-DNA relatedness (>70%), chemotaxonomic, and molecular profile, P. pseudoalcaligenes ATCC 17440(T) and P. oleovorans ATCC 8062(T) should be united under the same name; according to the rules of priority, P. oleovorans, the first described species, is the earlier synonym and P. pseudoalcaligenes is the later synonym. As a consequence, the division of the species P. oleovorans into two novel subspecies is proposed: P. oleovorans subsp. oleovorans subsp. nov. (type strain ATCC 8062(T) = DSM 1045(T) = NCIB 6576(T)), P. oleovorans subsp. lubricantis subsp. nov. (type strain RS1(T) = ATCC BAA-1494(T) = DSM 21016(T)). PMID:19936829

  5. ATCC 43642 replaces ATCC 23581 as the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926. Opinion 91. Judicial Commission of the International Committee on Systematics of Prokaryotes.

    PubMed

    Tindall, B J

    2014-10-01

    The Judicial Commission affirms that, according to information presented to it, the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926 designated on the Approved Lists of Bacterial Names (ATCC 23581) has been shown not to represent an authentic culture of strain RGA (a member of the serovar Icterohaemorrhagiae) and ATCC 43642, derived from an authentic strain of strain RGA, a member of the serovar Icterohaemorrhagiae, is designated the type strain of Leptospira interrogans (Stimson 1907) Wenyon 1926.

  6. Growth of Lactobacillus paracasei ATCC334 in a cheese model system: A biochemical approach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Growth of Lactobacillus paracasei ATCC 334, in a cheese-ripening model system based upon a medium prepared from ripening Cheddar cheese extract (CCE) was evaluated. Lactobacillus paracasei ATCC 334 grows in CCE made from cheese ripened for 2 (2mCCE), 6 (6mCCE), and 8 (8mCCE) mo, to final cell densit...

  7. Complete Genome Sequence of Arthrobacter sp. ATCC 21022, a Host for Bacteriophage Discovery

    PubMed Central

    Russell, Daniel A.

    2016-01-01

    We report the complete genome sequence of Arthrobacter sp. ATCC 21022, a strain maintained by ATCC and a commonly used host for bacteriophage isolation and genomic analysis. The strain is prophage-free and CRISPR-free but codes for two predicted restriction-modification systems. PMID:27013048

  8. Identification of the Herboxidiene Biosynthetic Gene Cluster in Streptomyces chromofuscus ATCC 49982

    PubMed Central

    Shao, Lei; Zi, Jiachen; Zeng, Jia

    2012-01-01

    The 53-kb biosynthetic gene cluster for the novel anticholesterol natural product herboxidiene was identified in Streptomyces chromofuscus ATCC 49982 by genome sequencing and gene inactivation. In addition to herboxidiene, a biosynthetic intermediate, 18-deoxy-herboxidiene, was also isolated from the fermentation broth of S. chromofuscus ATCC 49982 as a minor metabolite. PMID:22247174

  9. Complete Genome Sequence of the Type Strain of Aeromonas schubertii, ATCC 43700

    PubMed Central

    Liu, Lihui; Zhang, Defeng; Fu, Xiaozhe; Shi, Cunbin; Lin, Qiang

    2016-01-01

    We sequenced the complete genome of the type strain of Aeromonas schubertii, ATCC 43700. The full genome sequence of A. schubertii ATCC 43700 is 4,356,858 bp, which encodes 3,842 proteins and contains 110 predicted RNA genes. PMID:26893413

  10. A Fruiting Body Tissue Method for Efficient Agrobacterium-Mediated Transformation of Agaricus bisporus

    PubMed Central

    Chen, Xi; Stone, Michelle; Schlagnhaufer, Carl; Romaine, C. Peter

    2000-01-01

    We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species. PMID:11010906

  11. Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications.

    PubMed

    Krenek, Pavel; Samajova, Olga; Luptovciak, Ivan; Doskocilova, Anna; Komis, George; Samaj, Jozef

    2015-11-01

    Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations.

  12. Thermostable Cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073▿

    PubMed Central

    Li, Qingyan; Seffernick, Jennifer L.; Sadowsky, Michael J.; Wackett, Lawrence P.

    2009-01-01

    Cyanuric acid, a metabolic intermediate in the degradation of many s-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile Moorella thermoacetica ATCC 39073 was cloned, expressed in Escherichia coli, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and N-methylisocyanuric acid. The M. thermoacetica enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from M. thermoacetica is the most promising enzyme to use for cyanuric acid remediation applications. PMID:19767460

  13. Complete genome sequence of Anabaena variabilis ATCC 29413

    SciTech Connect

    Thiel, Teresa; Pratte, Brenda S.; Zhong, Jinshun; Goodwin, Lynne A.; Copeland, A; Lucas, Susan; Han, Cliff; Pitluck, Sam; Land, Miriam L; Kyrpides, Nikos C; Woyke, Tanja

    2013-01-01

    Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Ana-baena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40 C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence.

  14. Complete genome sequence of Anabaena variabilis ATCC 29413.

    PubMed

    Thiel, Teresa; Pratte, Brenda S; Zhong, Jinshun; Goodwin, Lynne; Copeland, Alex; Lucas, Susan; Han, Cliff; Pitluck, Sam; Land, Miriam L; Kyrpides, Nikos C; Woyke, Tanja

    2014-06-15

    Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Anabaena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40(°) C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence. PMID:25197444

  15. Biotransformation of (-)beta-pinene by Aspergillus niger ATCC 9642.

    PubMed

    Toniazzo, Geciane; de Oliveira, Débora; Dariva, Cláudio; Oestreicher, Enrique Guillermo; Antunes, Octávio A C

    2005-01-01

    The main objective of this work was to investigate the biotransformations of (-)alpha-pinene, (-)beta-pinene, and (+) limonene by Aspergillus niger ATCC 9642. The culture conditions involved--concentration of cosolvent (EtOH), substrate applied, and sequential addition of substrates were--investigated. Adaptation of the precultures with small amounts of substrate was also studied. The experiments were performed in conical flasks with liquid cultures. This strain of A. niger was able to convert only (-)beta-pinene into alpha-terpineol. An optimum conversion of (-)beta-pinene into alpha-terpineol of about 4% was obtained when the substrate was applied as a diluted solution in EtOH and sequential addition of substrate was used.

  16. Characterization of Agrobacterium tumefaciens DNA ligases C and D

    PubMed Central

    Zhu, Hui; Shuman, Stewart

    2007-01-01

    Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3′-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3′-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3′-OH end. The PE domains also have a 3′-phosphatase activity on an all-DNA primer-template that yields a 3′-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ. PMID:17488851

  17. Linear Chromosome-generating System of Agrobacterium tumefaciens C58

    PubMed Central

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood

    2012-01-01

    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide. PMID:22582388

  18. Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer.

    PubMed

    Pickardt, T; Meixner, M; Schade, V; Schieder, O

    1991-02-01

    Shoot tips and epicotyl-segments of Vicia narbonensis were co-cultivated with Agrobacterium tumefaciens strain C58C1 pGV 3850 HPT, carrying a plasmid coding for hygromycin-phosphotransferase. On callus-induction medium containing 60 mg/l hygromycin for selection, approximately 18% of the explants produced hygromycin-resistant callus. After transfer to regeneration-medium these calluses produced hygromycin-resistant and nopaline-positive somatic embryos which could be regenerated to plantlets. The integration of the T-DNA into the plant genome was confirmed by Southern analysis.

  19. Agrobacterium radiobacter and CDC group Ve-2 bacteremia.

    PubMed

    Blumberg, D A; Cherry, J D

    1989-01-01

    Agrobacterium radiobacter and CDC Group Ve-2 are rare human pathogens. The simultaneous infection with both of these bacteria in an immunocompromised host is reported. Review of the UCLA microbiology laboratory records revealed one additional case of A. radiobacter bacteremia and two additional cases of CDC Group Ve-2 bacteremia over a 3-year period. The clinical experience with these organisms is reviewed. Both organisms are opportunistic pathogens with a predilection for patients with foreign bodies in place. Although CDC Group Ve-2 bacteremia may respond to antibiotic therapy alone, the cure of A. radiobacter infections often requires foreign body removal.

  20. Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper.

    PubMed

    Karthikeyan, A S; Sarma, K S; Veluthambi, K

    1996-01-01

    Transformed Vigna mungo (blackgram) calli were obtained by cocultivating segments of primary leaves with Agrobacterium tumefaciens vir helper strains harbouring the binary vector pGA472 having kanamycin resistance gene as plant transformation marker. Transformed calli were selected on Murashige and Skoog medium supplemented with 50 mg/l kanamycin and 500 mg/l carbenicillin. Transformed calli were found to be resistant to kanamycin up to 900 mg/l concentration. Expression of kanamycin resistance gene in transformed calli was demonstrated by neomycin phosphotransferase assay. Stable integration of transferred DNA into V. mungo genome was confirmed by Southern blot analysis.

  1. Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare.

    PubMed

    Habibi, Peyman; de Sa, Maria Fatima Grossi; da Silva, André Luís Lopes; Makhzoum, Abdullah; da Luz Costa, Jefferson; Borghetti, Ivo Albertto; Soccol, Carlos Ricardo

    2016-04-01

    Origanum vulgare L is commonly known as a wild marjoram and winter sweet which has been used in the traditional medicine due to its therapeutic effects as stimulant, anticancer, antioxidant, antibacterial, anti-inflammatory and many other diseases. A reliable gene transfer system via Agrobacterium rhizogenes and plant regeneration via hairy roots was established in O. vulgare for the first time. The frequency of induced hairy roots was different by modification of the co-cultivation medium elements after infection by Agrobacterium rhizogenes strains K599 and ATCC15834. High transformation frequency (91.3 %) was achieved by co-cultivation of explants with A. rhizogenes on modified (MS) medium. The frequency of calli induction with an 81.5 % was achieved from hairy roots on MS medium with 0.25 mg/L(-1) 2,4-D. For shoot induction, initiated calli was transferred into a medium containing various concentrations of BA (0.1, 0.25, 0.5, 0.75 and 1 mg/L(-1)). The frequency of shoot generation (85.18 %) was achieved in medium fortified with 0.25 mg/L(-1) of BA. Shoots were placed on MS medium with 0.25 mg/l IBA for root induction. Roots appeared and induction rate was achieved after 15 days. PMID:27436918

  2. Metabolic changes in Agrobacterium tumefaciens-infected Brassica rapa.

    PubMed

    Simoh, Sanimah; Quintana, Naira; Kim, Hye Kyong; Choi, Young Hae; Verpoorte, Robert

    2009-07-01

    Agrobacterium has the ability to transfer its genetic material, T-DNA, into the plant genome. The unique interaction between the bacterium and its host plant has been well studied at the transcriptome, but not at the metabolic level. For a better understanding of this interaction it is necessary to investigate the metabolic changes of the host plant upon infection with Agrobacterium tumefaciens. This study investigated the metabolic response of Brassica rapa to infection with disarmed and tumor-inducing strains of A. tumefaciens using (1)H nuclear magnetic resonance spectroscopy combined with multivariate data analysis. The partial least square-discriminant analysis (PLS-DA) of two varieties of B. rapa showed that there was a clear differentiation in the metabolite profiles of B. rapa leaves infected with the disarmed strain LBA4404 and with tumor-inducing octopine and nopaline strains, particularly in the flavonoid, phenylpropanoid, sugar and free amino/organic acid contents. However, individual PLS-DA of each type of infection suggests that, in general, some flavonoids and phenylpropanoids were suppressed as a consequence of these infections. The results obtained in this study indicate that the disarmed strain LBA4404 and tumor-inducing strains have different effects on the metabolite profile of B. rapa.

  3. ACC deaminase activity in avirulent Agrobacterium tumefaciens D3.

    PubMed

    Hao, Youai; Charles, Trevor C; Glick, Bernard R

    2011-04-01

    Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and α-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58. PMID:21491979

  4. An Agrobacterium catalase is a virulence factor involved in tumorigenesis.

    PubMed

    Xu, X Q; Pan, S Q

    2000-01-01

    Most plant pathogenic bacteria adopt the type III secretion systems to secrete virulence factors and/or avirulence gene products, which trigger the plant hypersensitive response (HR) and the oxidative burst with hydrogen peroxide (H2O2) as the main component. However, the soil-borne plant pathogen Agrobacterium tumefaciens uses the type IV secretion pathway to deliver its oncogenic T-DNA that causes crown gall tumours on many plant species. A. tumefaciens does not elicit a typical HR on those plants. Here, we report that inactivation of one of A. tumefaciens catalases (which converts H2O2 to H2O and O2) by a transposon insertion highly attenuated the bacterial ability to cause tumours on plants and to tolerate H2O2 toxicity, but not the bacterial viability in the absence of exogenous H2O2. This provides the first genetic evidence that the Agrobacterium-plant interaction involves a plant defence response, such as H2O2 production, and that catalase is a virulence factor for a plant pathogen. PMID:10652101

  5. Fate of Agrobacterium radiobacter K84 in the environment.

    PubMed Central

    Stockwell, V O; Moore, L W; Loper, J E

    1993-01-01

    Agrobacterium radiobacter K84 is an effective, commercially applied, biological control agent for the plant disease crown gall, yet little is known about the survival and dissemination of K84. To trace K84 in the environment, spontaneous antibiotic-resistant mutants were used. Growth rates and phenotypes of streptomycin- or rifampin-resistant K84 were similar to those of the parental K84, except the rifampin-resistant mutant produced less agrocin 84 as determined by bioassay. K84 and a strain of Agrobacterium tumefaciens established populations averaging 10(5) CFU/g in the rhizosphere of cherry and persisted on roots for 2 years. K84 established rhizosphere populations between 10(4) and 10(6) CFU/g on cherry, ryegrass, and 11 other herbaceous plants. Populations of K84 declined substantially in fallow soil or water over a 16-week period. K84 was detected in the rhizosphere of ryegrass located up to 40 cm from an inoculum source, indicating lateral dissemination of K84 in soil. In gall tissue on cherry, K84 established populations of 10(5) CFU/g, about 10- to 100-fold less than that of the pathogen. These data demonstrate that K84 persists for up to 2 years in a field environment as a rhizosphere inhabitant or in association with crown gall tissue. PMID:8357247

  6. Mutants of Agrobacterium tumefaciens with elevated vir gene expression

    SciTech Connect

    Pazour, G.J.; Ta, C.N.; Das, A. )

    1991-08-15

    Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG.

  7. Conjugation in Agrobacterium tumefaciens in the absence of plant tissue.

    PubMed Central

    Levin, R A; Farrand, S K; Gordon, M P; Nester, E W

    1976-01-01

    A general, reliable conjugation system for Agrobacterium tumefaciens in the absence of plant tissue is described in which A. tumefaciens can serve either as the donor or recipient of plasmid deoxyribonucleic acid with reasonable efficiency. Plasmid RP4 was transferred from Escherichia coli to A. tumefaciens and from strain of A. tumefaciens. Both RP4 and the A. tumefaciens virulence-associated plasmids were detected by alkaline sucrose gradients in A. tumefaciens strains A6 and C58 after mating with E. coli J53(RP4). The pathogenicity (tumor foramtion) of strains A6 and C58 and the sensitivity of strain C58 to bacteriocin 84 were unaffected by the acquistion of RP4 by the Agrobacterium strains. Plasmid R1drd-19 was not transferred to A. tumefaciens. Transformation experiments with plasmid deoxyribonucleic acid were unsuccessful, even though, in the case of RP4, conjugation studies showed taht the deoxyribonucleic acid was compatible with that of the recipient strains. PMID:783141

  8. Efficient method for Agrobacterium mediated transformation of Artemisia annua L.

    PubMed

    Alam, Pravej; Mohammad, Anis; Ahmad, M M; Khan, Mather Ali; Nadeem, Mohd; Khan, Riyazuddeen; Akmal, Mohd; Ahlawat, Seema; Abdin, M Z

    2014-01-01

    Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patents have been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidences of drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generate transgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laborious and delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediated transformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performance of T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times and co-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 under desiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated that the optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible.

  9. Biolistic- and Agrobacterium-mediated transformation protocols for wheat.

    PubMed

    Tamás-Nyitrai, Cecília; Jones, Huw D; Tamás, László

    2012-01-01

    After rice, wheat is considered to be the most important world food crop, and the demand for high-quality wheat flour is increasing. Although there are no GM varieties currently grown, wheat is an important target for biotechnology, and we anticipate that GM wheat will be commercially available in 10-15 years. In this chapter, we summarize the main features and challenges of wheat transformation and then describe detailed protocols for the production of transgenic wheat plants both by biolistic and Agrobacterium-mediated DNA-delivery. Although these methods are used mainly for bread wheat (Triticum aestivum L.), they can also be successfully applied, with slight modifications, to tetraploid durum wheat (T. turgidum L. var. durum). The appropriate size and developmental stage of explants (immature embryo-derived scutella), the conditions to produce embryogenic callus tissues, and the methods to regenerate transgenic plants under increasing selection pressure are provided in the protocol. To illustrate the application of herbicide selection system, we have chosen to describe the use of the plasmid pAHC25 for biolistic transformation, while for Agrobacterium-mediated transformation the binary vector pAL156 (incorporating both the bar gene and the uidA gene) has been chosen. Beside the step-by-step methodology for obtaining stably transformed and normal fertile plants, procedures for screening and testing transgenic wheat plants are also discussed.

  10. Genome Sequence of the Ethanol-Producing Zymomonas mobilis subsp. mobilis Lectotype Strain ATCC 10988 ▿

    PubMed Central

    Pappas, Katherine M.; Kouvelis, Vassili N.; Saunders, Elizabeth; Brettin, Thomas S.; Bruce, David; Detter, Chris; Balakireva, Mariya; Han, Cliff S.; Savvakis, Giannis; Kyrpides, Nikos C.; Typas, Milton A.

    2011-01-01

    Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome. PMID:21725006

  11. Dielectric characterization of forespores isolated from Bacillus megaterium ATCC 19213.

    PubMed Central

    Marquis, R E; Bender, G R; Carstensen, E L; Child, S Z

    1983-01-01

    Isolated stage III forespores of Bacillus megaterium ATCC 19213 in aqueous suspensions were nearly as dehydrated as mature spores, as indicated by low dextran-impermeable volumes of ca. 3.0 ml per g (dry weight) of cells compared with values of ca. 2.6 for mature spores and 7.3 for vegetative cells. The forespores lacked dipicolinate, had only minimal levels of calcium, magnesium, manganese, potassium, and sodium, and were more heat sensitive than vegetative cells. The effective homogeneous conductivities and dielectric constants measured over a frequency range of 1 to 200 MHz indicated that the inherent conductivities of the forespores were unusually low, in keeping with their low mineral contents, but that the forespores could be invaded by environmental ions which could penetrate dielectrically effective membranes. Overall, our findings support the view that the dehydration of a forespore during stage III of sporogenesis may be the result of ion movements out of the forespore into the sporangium. PMID:6401285

  12. Microcalorimetric study of cellulose degradation by Cellulomonas uda ATCC 21399

    SciTech Connect

    Dermoun, Z.; Belaich, J.P.

    1985-07-01

    A newly designed batch calorimeter was used to investigate the degradability of some celluloses having varying degrees of crystallinity. The PTC of an aerobic culture of Cellulomonas uda ATCC 21399 obtained revealed a diauxic growth which is attributed to the presence of hemicellulose contaminating Avicel and MN300 cellulose. The microcrystalline celluloses used were not completely utilized, whereas amorphous cellulose was easily metabolized, indicating that under the growth conditions used here, the physical structure of cellulose strongly influenced its microbial degradability. An equivalent growth yield of ca. 0.44 g/g was found with all the substrates used. The heat evolved by metabolism of one g cellulose was - 5.86 kJ/g, a value similar to that obtained with glucose culture. The growth rate was the only variable parameter. The data obtained showed as expected that the hydrolysis product of cellulose was consumed in the same way as that of glucose and that the only limiting factor to the biodegradability of cellulose was the breakdown of the polymeric substrate. It is concluded that data obtained with glucose metabolism can be used to evaluate the extent of cellulose degradation.

  13. L-Lactic Acid Production by Lactobacillus rhamnosus ATCC 10863

    PubMed Central

    Senedese, Ana Lívia Chemeli; Maciel Filho, Rubens; Maciel, Maria Regina Wolf

    2015-01-01

    Lactic acid has been shown to have the most promising application in biomaterials as poly(lactic acid). L. rhamnosus ATCC 10863 that produces L-lactic acid was used to perform the fermentation and molasses was used as substrate. A solution containing 27.6 g/L of sucrose (main composition of molasses) and 3.0 g/L of yeast extract was prepared, considering the final volume of 3,571 mL (14.0% (v/v) inoculum). Batch and fed batch fermentations were performed with temperature of 43.4°C and pH of 5.0. At the fed batch, three molasses feed were applied at 12, 24, and 36 hours. Samples were taken every two hours and the amounts of lactic acid, sucrose, glucose, and fructose were determined by HPLC. The sucrose was barely consumed at both processes; otherwise the glucose and fructose were almost entirely consumed. 16.5 g/L of lactic acid was produced at batch and 22.0 g/L at fed batch. Considering that lactic acid was produced due to the low concentration of the well consumed sugars, the final amount was considerable. The cell growth was checked and no substrate inhibition was observed. A sucrose molasses hydrolysis is suggested to better avail the molasses fermentation with this strain, surely increasing the L-lactic acid. PMID:25922852

  14. New insights into chloramphenicol biosynthesis in Streptomyces venezuelae ATCC 10712.

    PubMed

    Fernández-Martínez, Lorena T; Borsetto, Chiara; Gomez-Escribano, Juan Pablo; Bibb, Maureen J; Al-Bassam, Mahmoud M; Chandra, Govind; Bibb, Mervyn J

    2014-12-01

    Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916-sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na(+)/H(+) antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway.

  15. New Insights into Chloramphenicol Biosynthesis in Streptomyces venezuelae ATCC 10712

    PubMed Central

    Fernández-Martínez, Lorena T.; Borsetto, Chiara; Gomez-Escribano, Juan Pablo; Bibb, Maureen J.; Al-Bassam, Mahmoud M.; Chandra, Govind

    2014-01-01

    Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916–sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na+/H+ antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway. PMID:25267678

  16. Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production.

    PubMed

    Lin, Jianping; Fu, Weiqi; Cen, Peilin

    2009-04-01

    The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and D-glucose, D-fructose, D-xylose, D-mannose, L-arabinose, D-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20mM D-glucose or D-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining D-xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3g/l (56 mM) under the appropriate conditions in the fermenter.

  17. Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infiltration of tobacco leaves with a suspension of Agrobacterium tumefaciens harboring a binary plant expression plasmid provides a convenient method for laboratory scale protein production. When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana), diffic...

  18. Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens.

    PubMed

    Radke, S E; Turner, J C; Facciotti, D

    1992-09-01

    Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1-9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus.

  19. Mechanisms and regulation of polar surface attachment in Agrobacterium tumefaciens.

    PubMed

    Tomlinson, Amelia D; Fuqua, Clay

    2009-12-01

    Agrobacterium tumefaciens is a plant pathogen that transfers a segment of its own DNA into host plants to cause Crown Gall disease. The infection process requires intimate contact between the infecting bacteria and the host tissue. A. tumefaciens attaches efficiently to plant tissues and to abiotic surfaces, and can establish complex biofilms at colonization sites. The dominant mode of attachment is via a single pole in contact with the surface. Several different appendages, adhesins and adhesives play roles during attachment, and foster the transition from free-swimming to sessile growth. This polar surface interaction reflects a more fundamental cellular asymmetry in A. tumefaciens that influences and is congruent with its attached lifestyle. PMID:19879182

  20. Corn metabolites affect growth and virulence of Agrobacterium tumefaciens.

    PubMed Central

    Sahi, S V; Chilton, M D; Chilton, W S

    1990-01-01

    Homogenates of corn seedlings inhibit both growth of Agrobacterium tumefaciens and induction of its Ti plasmid virulence (vir) genes by acetosyringone (AS). The heat-labile inhibitor has been identified as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), present in 2-week-old seedlings (B73) at a concentration of 1.5 mM or greater. A concentration of 0.3 mM DIMBOA is sufficient to block growth of A. tumefaciens completely for 220 hr. DIMBOA at 0.1 mM concentration completely inhibited vir gene induction by 100 microM AS and reduced growth rate by 50%. Thus, DIMBOA can be expected to have a significant effect on attempts to transform corn by using A. tumefaciens as a vector. Images PMID:11607078

  1. Agrobacterium tumefaciens-mediated transformation of Botryosphaeria dothidea.

    PubMed

    Chen, Liang; Wang, Qun; Chen, Hua; Sun, Gengwu; Liu, Huixiang; Wang, Hongkai

    2016-07-01

    Botryosphaeria dothidea is a severe causal agent of die-back and cankers of many woody plants and causes great losses in many regions. The pathogenic mechanism of this pathogen has not been well explored due to lack of mutants and genetic information. In this study, we developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for B. dothidea protoplasts using vector pBHt2 containing the hph gene as a selection marker under the control of trp C promoter. Using this protocol we successfully generated the B. dothidea transformants with efficiency about 23 transformants per 10(5) protoplasts. This is the first report of genetic transformation of B. dothidea via ATMT and this protocol provides an effective tool for B. dothidea genome manipulation, gene identification and functional analysis. PMID:27263001

  2. Agrobacterium-mediated transformation of Ruta graveolens L.

    PubMed

    Lièvre, Karine; Tran, Thi Lê Minh; Doerper, Sébastien; Hehn, Alain; Lacoste, Paul; Thomasset, Brigitte; Bourgaud, Frédéric; Gontier, Eric

    2009-01-01

    Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.

  3. Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens.

    PubMed

    Radke, S E; Turner, J C; Facciotti, D

    1992-09-01

    Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1-9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus. PMID:24213157

  4. Toxicity and mutagenesis of chrysotile asbestos to Agrobacterium radiobacter.

    PubMed

    Yoshida, N; Naka, T; Sengoku, T; Ogawa, K

    2001-06-01

    The mutation of Agrobacterium radiobacter cells exposed to chrysotile asbestos was examined by the random amplified polymorphic DNA (RAPD) method. Approximately 1.4 kbp of DNA in A. radiobacter, which was not amplified strongly in the cells that were not exposed to asbestos, was amplified in the cells that were exposed to asbestos. Mutation in genomic DNA of A. radiobacter was found to be induced by asbestos. Specific DNA that was amplified by asbestos present in PCR products and that which exists latently in genomic DNA were cloned, and these sequences were then determined and compared. It was shown that one of the mutations by the asbestos in the A. radiobacter occurred only in the primer annealed region and was a point mutation or deletion.

  5. Efficient production of transgenic melon via Agrobacterium-mediated transformation.

    PubMed

    Bezirganoglu, I; Hwang, S Y; Shaw, J F; Fang, T J

    2014-04-25

    Oriental melon (Cucumis melo L. var. makuwa) is an important fruit for human consumption. However, this plant species is one of the most recalcitrant to genetic transformation. The lack of an efficient in vitro system limits the development of a reproducible genetic transformation protocol for Oriental melon. In this study, an efficient transgenic production method for Agrobacterium-mediated transformation using cotyledon explants of Oriental melon was developed. Cotyledon explants were pre-cultivated for two days in the dark, and the optimal conditions for transformation of melon were determined to be a bacteria concentration of OD600 0.6, inoculation for 30 min, and two days of co-cultivation. Transgenic melon plants were produced from kanamycin-resistant shoots. A total of 11 independent transgenic plants were regenerated with a transformation efficiency of 0.8% of the inoculated explants. The transgenic plants were phenotypically normal and fully fertile, which might be a consequence of the co-cultivation time.

  6. Progress of cereal transformation technology mediated by Agrobacterium tumefaciens.

    PubMed

    Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko

    2014-01-01

    Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens. PMID:25426132

  7. Progress of cereal transformation technology mediated by Agrobacterium tumefaciens

    PubMed Central

    Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko

    2014-01-01

    Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens. PMID:25426132

  8. Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species).

    PubMed

    Prasad, Binod; Vadakedath, Nithya; Jeong, Hyun-Jeong; General, Thiyam; Cho, Man-Gi; Lein, Wolfgang

    2014-10-01

    Isochrysis galbana and Isochrysis sp. are economically important microalgae from the division of haptophytes. Here, we report Agrobacterium-mediated stable DNA transfer into their nuclear genomes. Initial studies were performed to standardize co-cultivation media and determine the sensitivity of the microalgae to selective agents. Up to 1 mg/ml of the antibiotic hygromycin did not inhibit growth, whereas both the haptophytes bleached in artificial seawater (ASW) medium containing micromolar concentrations of the herbicide norflurazon. Co-cultivation of Isochrysis sp. and I. galbana with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1380-pds-L504R yielded norflurazon-resistant (NR) colonies visible on selective plates after 20-30 days. pCAMBIA 1380-pds-L540R was constructed by cloning a mutated genomic phytoene desaturase (pds) gene from Haematococcus pluvialis as a selectable marker gene into the binary vector system pCAMBIA 1380. Co-cultivation of Isochrysis sp. with A. tumefaciens in ASW medium containing 200 μM of acetosyringone for 72 h produced the highest number of NR cells. For I. galbana, 100 μM of acetosyringone, ASW medium, and 48 h co-cultivation period appeared to be optimum co-cultivation parameters. The NR colonies kept their resistance phenotype for at least 24 months, even in the absence of selective pressure. The transfer of the pds gene in NR cells was shown by PCR amplification of the T-DNA sequences from the genomic DNA of NR cells and Southern blot analysis using T-DNA sequences as probes. The genetic manipulation described here will allow metabolic engineering and a better understanding of several biochemical pathways in the future.

  9. Attachment of Agrobacterium tumefaciens B6 and A. radiobacter K84 to Tomato Root Tips

    PubMed Central

    Penalver, R.; Serra, M. T.; Duran-Vila, N.; Lopez, M. M.

    1996-01-01

    Agrobacterium tumefaciens B6 and the avirulent Agrobacterium radiobacter strain K84 attached to in vitro-cultured tomato root tips, but the binding of strain B6 to root tips was greater than the binding of strain K84. Strain K84 was not able to block the attachment of A. tumefaciens B6 to in vitro-cultured tomato root tips. PMID:16535413

  10. vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens.

    PubMed Central

    Gelvin, S B; Habeck, L L

    1990-01-01

    Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiR10 was found to cause a 100- to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPH1JI was not impaired in any of the mutant Agrobacterium strains tested. The effect of vir gene function on the conjugal transfer of the Ti plasmid suggests that a relationship may exist between the processes that control the transfer of the T-DNA from Agrobacterium to plant cells and the conjugal transfer of the Ti plasmid between bacterial cells. PMID:2155206

  11. Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens

    PubMed Central

    Zhou, Xiaohong; Wang, Ke; Lv, Dongwen; Wu, Chengjun; Li, Jiarui; Zhao, Pei; Lin, Zhishan; Du, Lipu; Yan, Yueming; Ye, Xingguo

    2013-01-01

    Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops. PMID:24278131

  12. Arabidopsis VIRE2 INTERACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants.

    PubMed

    Anand, Ajith; Krichevsky, Alexander; Schornack, Sebastian; Lahaye, Thomas; Tzfira, Tzvi; Tang, Yuhong; Citovsky, Vitaly; Mysore, Kirankumar S

    2007-05-01

    Agrobacterium tumefaciens-mediated genetic transformation is an efficient tool for genetic engineering of plants. VirE2 is a single-stranded DNA binding Agrobacterium protein that is transported into the plant cell and presumably protects the T-DNA from degradation. Using a yeast two-hybrid system, we identified Arabidopsis thaliana VIRE2-INTERACTING PROTEIN2 (VIP2) with a NOT domain that is conserved in both plants and animals. Furthermore, we provide evidence supporting VIP2 interaction with VIP1, a basic domain/leucine zipper motif-containing protein required for nuclear import and integration of T-DNA. Virus-induced gene silencing of VIP2 in Nicotiana benthamiana and characterization of the Arabidopsis vip2 mutant (At vip2) demonstrate that VIP2 is required for Agrobacterium-mediated stable transformation but not for transient transformation. Assays based upon a promoter-trap vector and quantification of T-DNA integration further confirmed VIP2 involvement in T-DNA integration. Interestingly, VIP2 transcripts were induced to a greater extent over prolonged periods after infection with a T-DNA transfer-competent Agrobacterium strain compared with the transfer-deficient Agrobacterium strain. Transcriptome analyses of At vip2 suggest that VIP2 is likely a transcriptional regulator, and the recalcitrancy to transformation in At vip2 is probably due to the combination of muted gene expression response upon Agrobacterium infection and repression of histone genes resulting in decreased T-DNA integration events. PMID:17496122

  13. Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

    PubMed

    Srinivasan, Ramachandran; Gothandam, Kodiveri Muthukalianan

    2016-01-01

    An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer. PMID:27351975

  14. Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination.

    PubMed

    Mishiba, Kei-ichiro; Chin, Dong Poh; Mii, Masahiro

    2005-07-01

    A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both beta-glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3-1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l(-1) hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.

  15. Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

    PubMed

    Srinivasan, Ramachandran; Gothandam, Kodiveri Muthukalianan

    2016-01-01

    An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

  16. Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.

    PubMed

    Zhang, Wan-Jun; Dewey, Ralph E; Boss, Wendy; Phillippy, Brian Q; Qu, Rongda

    2013-02-01

    Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.

  17. Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation

    PubMed Central

    Srinivasan, Ramachandran

    2016-01-01

    An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer. PMID:27351975

  18. Colonization and immunomodulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract.

    PubMed

    Valeur, Nana; Engel, Peter; Carbajal, Noris; Connolly, Eamonn; Ladefoged, Karin

    2004-02-01

    Lactobacillus reuteri ATCC 55730 is a probiotic (health-promoting) bacterium widely used as a dietary supplement. This study was designed to examine local colonization of the human gastrointestinal mucosa after dietary supplementation with L. reuteri ATCC 55730 and to determine subsequent immune responses at the colonized sites. In this open clinical investigation, 10 healthy volunteers and 9 volunteers with ileostomy underwent gastroscopy or ileoscopy and biopsy samples were taken from the stomach, duodenum, or ileum before and after supplementation with 4 x 10(8) CFU of live L. reuteri ATCC 55730 lactobacilli per day for 28 days. Biopsy specimen colonization was analyzed using fluorescence in situ hybridization with a molecular beacon probe, and immune cell populations were determined by immunostaining. Endogenous L. reuteri was detected in the stomach of 1 subject and the duodenum of 3 subjects (out of 10 subjects). After L. reuteri ATCC 55730 supplementation, the stomachs of 8 and the duodenums of all 10 subjects were colonized. Three ileostomy subjects (of six tested) had endogenous L. reuteri at baseline, while all six displayed colonization after L. reuteri supplementation. Gastric mucosal histiocyte numbers were reduced and duodenal B-lymphocyte numbers were increased by L. reuteri ATCC 55730 administration. Furthermore, L. reuteri administration induced a significantly higher amount of CD4-positive T-lymphocytes in the ileal epithelium. Dietary supplementation with the probiotic L. reuteri ATCC 55730 induces significant colonization of the stomach, duodenum, and ileum of healthy humans, and this is associated with significant alterations of the immune response in the gastrointestinal mucosa. These responses may be key components of a mechanism by which L. reuteri ATCC 55730 exerts its well-documented probiotic effects in humans.

  19. Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation

    SciTech Connect

    McKeown, Catherine K; Brown, Steven D

    2011-01-01

    The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. A time-series analysis of gene expression revealed changes in transcript levels of {approx}40% of genes ({approx}1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligosaccharide hydrolysis products

  20. Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation

    PubMed Central

    2011-01-01

    Background The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. Results A time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Conclusions Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligosaccharide

  1. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  2. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H.C.

    1998-07-14

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  3. Fermentation of residual glycerol by Clostridium acetobutylicum ATCC 824 in pure and mixed cultures.

    PubMed

    Dams, Rosemeri I; Guilherme, Alexandre A; Vale, Maria S; Nunes, Vanja F; Leitão, Renato C; Santaella, Sandra T

    2016-12-01

    The aim of this research was to estimate the production of hydrogen, organic acids and alcohols by the strain of Clostridium acetobutylicum ATCC 824 using residual glycerol as a carbon source. The experiments were carried out in pure and mixed cultures in batch experiments. Three different sources of inocula for mixed culture were used. Ruminal liquid from goats and sludge collected from two upflow anaerobic sludge blanket reactors treating municipal wastewater and brewery effluent were tested for hydrogen, organic acids and alcohols production with or without C. acetobutylicum ATCC 824. The main detected end-products from the glycerol fermentation were hydrogen, organic acids (acetic, propionic, butyric and caproic) and alcohol (ethanol and 1,3-propanediol - 1,3PD). High hydrogen (0.44 mol H2/mol glycerol consumed) and 1,3PD (0.32 mol 1,3PD/mol glycerol consumed) yields were obtained when the strain C. acetobutylicum ATCC 824 was bioaugmented into the sludge from municipal wastewater using 5 g/L of glycerol. Significant concentrations of n-caproic acid were detected in the ruminal liquid when amended with C. acetobutylicum ATCC 824. The results suggest that glycerol can be used for the generation of H2, 1,3PD and n-caproic acid using C. acetobutylicum ATCC 824 as agent in pure or mixed cultures.

  4. Succinic Semialdehyde Promotes Prosurvival Capability of Agrobacterium tumefaciens

    PubMed Central

    Wang, Chao; Tang, Desong; Gao, Yong-Gui

    2016-01-01

    ABSTRACT Succinic semialdehyde (SSA), an important metabolite of γ-aminobutyric acid (GABA), is a ligand of the repressor AttJ regulating the expression of the attJ-attKLM gene cluster in the plant pathogen Agrobacterium tumefaciens. While the response of A. tumefaciens to GABA and the function of attKLM have been extensively studied, genetic and physiological responses of A. tumefaciens to SSA remain unknown. In combination with microarray and genetic approaches, this study sets out to explore new roles of the SSA-AttJKLM regulatory mechanism during bacterial infection. The results showed that SSA plays a key role in regulation of several bacterial activities, including C4-dicarboxylate utilization, nitrate assimilation, and resistance to oxidative stress. Interestingly, while the SSA relies heavily on the functional AttKLM in mediating nitrate assimilation and oxidative stress resistance, the compound could regulate utilization of C4-dicarboxylates independent of AttJKLM. We further provide evidence that SSA controls C4-dicarboxylate utilization through induction of an SSA importer and that disruption of attKLM attenuates the tumorigenicity of A. tumefaciens. Taken together, these findings indicate that SSA could be a potent plant signal which, together with AttKLM, plays a vital role in promoting the bacterial prosurvival abilities during infection. IMPORTANCE Agrobacterium tumefaciens is a plant pathogen causing crown gall diseases and has been well known as a powerful tool for plant genetic engineering. During the long history of microbe-host interaction, A. tumefaciens has evolved the capabilities of recognition and response to plant-derived chemical metabolites. Succinic semialdehyde (SSA) is one such metabolite. Previous results have demonstrated that SSA functions to activate a quorum-quenching mechanism and thus to decrease the level of quorum-sensing signals, thereby avoiding the elicitation of a plant defense. Here, we studied the effect of SSA on gene

  5. Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac- to Lac+.

    PubMed

    Tsai, Yu-Kuo; Chen, Hung-Wen; Lo, Ta-Chun; Lin, Thy-Hou

    2009-03-01

    Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac(+) clones could be obtained. A gene cluster (lacTEGF-galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac(-)) and its Lac(+) revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac-gal gene clusters was different. The protein sequence identity between the lac-gal gene cluster and those reported previously for some L. casei (Lac(+)) strains was high; namely, 96-100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac(+) revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac(+) revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac(-) to Lac(+) for L. casei ATCC 27139. PMID:19246746

  6. Expression of Clostridium acetobutylicum ATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

    PubMed Central

    Bermejo, Lourdes L.; Welker, Neil E.; Papoutsakis, Eleftherios T.

    1998-01-01

    A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided. PMID:9501448

  7. Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

    PubMed Central

    Binda, Elisa; Carrano, Lucia; Bibb, Mervyn; Marinelli, Flavia

    2014-01-01

    Glycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the β-lactam-sensitive d,d-peptidase/d,d-carboxypeptidase encoded by vanYn, the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanYn gene dosage and expressed vanHatAatXat from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanYn, complementing a vanYn mutant, or duplicating vanYn had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYn activity. The heterologous expression of vanHatAatXat increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanYn-based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins. PMID:24957828

  8. Relationship between glycopeptide production and resistance in the actinomycete Nonomuraea sp. ATCC 39727.

    PubMed

    Marcone, Giorgia Letizia; Binda, Elisa; Carrano, Lucia; Bibb, Mervyn; Marinelli, Flavia

    2014-09-01

    Glycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the β-lactam-sensitive D,D-peptidase/D,D-carboxypeptidase encoded by vanYn, the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanYn gene dosage and expressed vanHatAatXat from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanYn, complementing a vanYn mutant, or duplicating vanYn had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYn activity. The heterologous expression of vanHatAatXat increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanYn-based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins.

  9. Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG.

    PubMed Central

    Pazour, G J; Ta, C N; Das, A

    1992-01-01

    The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmids are positively regulated by virG in conjunction with virA and plant-derived inducing molecules. A procedure that utilizes both genetic selection and a genetic screen was developed to isolate mutations in virG that led to elevated levels of vir gene expression in the absence of virA and plant phenolic inducers. Mutants were isolated at a frequency of 1 in 10(7) to 10(8). Substitution mutations at two positions in the virG coding region were found to result in the desired phenotype. One mutant had an asparagine-to-aspartic acid substitution at residue 54, and the other contained an isoleucine-to-leucine substitution at residue 106. In both cases, the mutant phenotype required the presence of the active-site aspartic acid residue at position 52. Further analysis showed that no other substitution at residue 54 resulted in a constitutive phenotype. In contrast, several substitutions at residue 106 led to a constitutive phenotype. The possible roles of the residues at positions 54 and 106 in VirG function are discussed. PMID:1597431

  10. Diversity among B6 strains of Agrobacterium tumefaciens.

    PubMed Central

    Hamada, S E; Farrand, S K

    1980-01-01

    A total of 20 laboratory substrains of Agrobacterium tumefaciens strain B6 were compared with respect to six characteristics, including 3-ketolactose production, lysogeny, octopine catabolism, tumorigenic host range, and plasmid content. Within this group of strains diversity was found for all characteristics except 3-ketolactose production. Six substrains were lysogenized with an omega-type phage, whereas one substrain appeared neither sensitive to nor lysogenized with this bacteriophage. All but two substrains catabolized octopine and induced tumors on carrot disks. These 18 substrains harbor deoxyribonucleic acid sequences homologous to pTiB6-806. The two substrains unable to catabolize octopine were nontumorigenic and lacked detectable Ti plasmid sequences. Of the 20 substrains, 13 also contained sequences homologous to the cryptic plasmid pAtB6-806; 2 of the 18 substrains tumorigenic on carrots failed to induce tumors on Kalanchoe leaves. Their inability to induced tumors on this host, could not be correlated with lysogeny, with the presence or absence of pAtB6-806, or with the very large cryptic plasmid recently described. The Ti plasmids from these two strains were indistinguishable from pTiB6-806 by restriction enzyme analysis and could genetically convert a cured A. tumefaciens strain to tumorigenicity on both plant species. The results with these two strains suggest that parameters of tumorigenicity, such as host range, may be controlled by the bacterial chromosome. Images PMID:7364725

  11. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens

    SciTech Connect

    Amikam, D.; Benziman, M. )

    1989-12-01

    The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of {sup 32}P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with ({sup 14}C)glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes.

  12. Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens.

    PubMed

    Czolkoss, Simon; Fritz, Christiane; Hölzl, Georg; Aktas, Meriyem

    2016-01-01

    Cardiolipin (CL) is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG) by phospholipase D-type cardiolipin synthases (PLD-type Cls). In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1) and atu2486 (cls2), coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants. PMID:27472399

  13. Functions and regulation of quorum-sensing in Agrobacterium tumefaciens

    PubMed Central

    Lang, Julien; Faure, Denis

    2014-01-01

    In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS). The determinants of the QS-system belong to the LuxR/LuxI class. The LuxI-like protein TraI synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last 20 years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/LuxI type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review, we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids. PMID:24550924

  14. Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.).

    PubMed Central

    Khan, MRI.; Tabe, L. M.; Heath, L. C.; Spencer, D.; Higgins, TJV.

    1994-01-01

    We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, [beta]-glucuronidase (GUS), neomycin phosphotransferase, and an [alpha]-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait. PMID:12232188

  15. Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens

    PubMed Central

    Czolkoss, Simon; Fritz, Christiane; Hölzl, Georg; Aktas, Meriyem

    2016-01-01

    Cardiolipin (CL) is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG) by phospholipase D-type cardiolipin synthases (PLD-type Cls). In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1) and atu2486 (cls2), coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants. PMID:27472399

  16. Biodegradation of Glycerol Trinitrate and Pentaerythritol Tetranitrate by Agrobacterium radiobacter

    PubMed Central

    White, G. F.; Snape, J. R.; Nicklin, S.

    1996-01-01

    Bacteria capable of metabolizing highly explosive and vasodilatory glycerol trinitrate (GTN) were isolated under aerobic and nitrogen-limiting conditions from soil, river water, and activated sewage sludge. One of these strains (from sewage sludge) chosen for further study was identified as Agrobacterium radiobacter subgroup B. A combination of high-pressure liquid chromatography and nuclear magnetic resonance analyses of the culture medium during the growth of A. radiobacter on basal salts-glycerol-GTN medium showed the sequential conversion of GTN to glycerol dinitrates and glycerol mononitrates. Isomeric glycerol 1,2-dinitrate and glycerol 1,3-dinitrate were produced simultaneously and concomitantly with the disappearance of GTN, with significant regioselectivity for the production of the 1,3-dinitrate. Dinitrates were further degraded to glycerol 1- and 2-mononitrates, but mononitrates were not biodegraded. Cells were also capable of metabolizing pentaerythritol tetranitrate, probably to its trinitrate and dinitrate analogs. Extracts of broth-grown cells contained an enzyme which in the presence of added NADH converted GTN stoichiometrically to nitrite and the mixture of glycerol dinitrates. The specific activity of this enzyme was increased 160-fold by growth on GTN as the sole source of nitrogen. PMID:16535244

  17. Maize (Zea mays L.) transformation by Agrobacterium tumefaciens infection of pollinated ovules.

    PubMed

    Chen, Liang; Cong, Yuanyuan; He, Hongxia; Yu, Ying

    2014-02-10

    A novel transformation system was established for maize using Agrobacterium infection of in vitro cultured ovules. The maize ovules were isolated 24h after pollination and infected with Agrobacterium. The embryos were isolated from the pollinated ovules 2-3 weeks after Agrobacterium infection, regenerated to plantlets and investigated for transgene expression and inheritance. Experimental evaluations were focused on the four main aspects. Firstly, through the introduction of gus gene for monitoring transformation and development of embryo, it was confirmed that transgenic plants can be generated from in vitro cultured maize ovules infected with Agrobacterium. Secondly, in order to standardize the transformation protocol, several important factors that affected transformation efficiency were optimized. They included Agrobacterium delivery approach, surfactant, AS concentration, and cocultivation duration. Thirdly, stable expression and Mendelian inheritance of the introduced genes were analyzed in independent lines over two generations. Fourthly, the pollinated ovule culture-regeneration potential and transformation efficiency of five maize inbred lines were investigated to confirm the genotype independence of this transformation system. We conclude that the transformation system established in this study can be used to generate high-quality transgenic maize plants rapidly and directly.

  18. Agrobacterium arsenijevicii sp. nov., isolated from crown gall tumors on raspberry and cherry plum.

    PubMed

    Kuzmanović, Nemanja; Puławska, Joanna; Prokić, Anđelka; Ivanović, Milan; Zlatković, Nevena; Jones, Jeffrey B; Obradović, Aleksa

    2015-09-01

    Two plant-tumorigenic strains KFB 330(T) and KFB 335 isolated from galls on raspberry (Rubus idaeus) in Serbia, and a non-pathogenic strain AL51.1 recovered from a cherry plum (Prunus cerasifera) tumor in Poland, were genotypically and phenotypically characterized. Phylogenetic reconstruction based on 16S rDNA placed them within the genus Agrobacterium, with A. nepotum as their closest relative. Multilocus sequence analysis (MLSA) based on the partial sequences of atpD, glnA, gyrB, recA and rpoB housekeeping genes suggested that these three strains represent a new Agrobacterium species, that clustered with type strains of A. nepotum, A. radiobacter, "A. fabrum" and A. pusense. This was further supported by average nucleotide identity values (<92%) between the whole genome sequences of strain KFB 330(T) and related Agrobacterium species. The major cellular fatty acids of the novel strains were 18:1 w7c (72.8-77.87%) and 16:0 (6.82-8.58%). Phenotypic features allowed their differentiation from closely related species. Polyphasic characterization showed that the three strains represent a novel species of the genus Agrobacterium, for which the name Agrobacterium arsenijevicii sp. nov. is proposed. The type strain of A. arsenijevicii is KFB 330(T) (= CFBP 8308(T) = LMG 28674(T)). PMID:26117193

  19. Agrobacterium arsenijevicii sp. nov., isolated from crown gall tumors on raspberry and cherry plum.

    PubMed

    Kuzmanović, Nemanja; Puławska, Joanna; Prokić, Anđelka; Ivanović, Milan; Zlatković, Nevena; Jones, Jeffrey B; Obradović, Aleksa

    2015-09-01

    Two plant-tumorigenic strains KFB 330(T) and KFB 335 isolated from galls on raspberry (Rubus idaeus) in Serbia, and a non-pathogenic strain AL51.1 recovered from a cherry plum (Prunus cerasifera) tumor in Poland, were genotypically and phenotypically characterized. Phylogenetic reconstruction based on 16S rDNA placed them within the genus Agrobacterium, with A. nepotum as their closest relative. Multilocus sequence analysis (MLSA) based on the partial sequences of atpD, glnA, gyrB, recA and rpoB housekeeping genes suggested that these three strains represent a new Agrobacterium species, that clustered with type strains of A. nepotum, A. radiobacter, "A. fabrum" and A. pusense. This was further supported by average nucleotide identity values (<92%) between the whole genome sequences of strain KFB 330(T) and related Agrobacterium species. The major cellular fatty acids of the novel strains were 18:1 w7c (72.8-77.87%) and 16:0 (6.82-8.58%). Phenotypic features allowed their differentiation from closely related species. Polyphasic characterization showed that the three strains represent a novel species of the genus Agrobacterium, for which the name Agrobacterium arsenijevicii sp. nov. is proposed. The type strain of A. arsenijevicii is KFB 330(T) (= CFBP 8308(T) = LMG 28674(T)).

  20. Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens.

    PubMed

    Wilms, Ina; Overlöper, Aaron; Nowrousian, Minou; Sharma, Cynthia M; Narberhaus, Franz

    2012-04-01

    Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium.

  1. CAMP test detected Staphylococcus delphini ATCC 49172 beta-haemolysin production.

    PubMed

    Savini, Vincenzo; Kosecka, Maja; Marrollo, Roberta; Carretto, Edoardo; Miedzobrodzki, Jacek

    2013-01-01

    Through a CAMP test, we first observed a Staphylococcus delphini strain (ATCC 49172) to release beta-haemolysin. Production of the latter in this coagulase-positive species of the 'Staphylococcus intermedius Group', in fact, has been labeled to be undetermined, thus far. Of course, a wider number of strains have to be investigated in order to define whether this property is constitutive (like in Staphylococcus (pseud)intermedius), or strain-dependent (like in Staphylococcus aureus), and which clinical impact it has; nevertheless, we can state that S. delphini ATCC 49172 indeed produces this toxin.

  2. Draft Genome Sequence of Agrobacterium sp. Strain R89-1, a Morphine Alkaloid-Biotransforming Bacterium

    PubMed Central

    Kyslíková, Eva

    2016-01-01

    Agrobacterium sp. strain R89-1 isolated from composted wastes of Papaver somniferum can effectively biotransform codeine/morphine into 14-OH-derivatives. Here, we present a 4.7-Mb assembly of the R89-1 strain genome. The draft shows that the strain R89-1 represents a distinct phylogenetic lineage within the genus Agrobacterium. PMID:27056219

  3. Effect of Agrobacterium culture and inoculation density on transformation efficiency of a citrange (Citrus reticulata x Poncirus trifoliata).

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of Agrobacterium growth phase and density on transformation of citrus rootstock US-812 (Citrus reticulata x Poncirus trifoliata) epicotyl explants was determined. In the first experiment, Agrobacterium EHA105 containing pBINGUSint was grown in YEP medium to an OD600 of 1 and glycerol sto...

  4. Draft Genome Sequence of Clostridium scatologenes ATCC 25775, a Chemolithoautotrophic Acetogenic Bacterium Producing 3-Methylindole and 4-Methylphenol

    PubMed Central

    Song, Yoseb; Jeong, Yujin; Shin, Hyeon Seok

    2014-01-01

    Clostridium scatologenes ATCC 25775 is a strictly anaerobic and chemolithoautotrophic acetogenic bacterium that converts syngas into multi-carbon compounds such as acetate, indole, 3-methylindole, and 4-methylphenol. Here we report the draft genome sequence of C. scatologenes ATCC 25775 (7.3 Mbp) to elucidate its metabolic pathway for syngas fermentation. PMID:24831152

  5. The role of RAR1 in Agrobacterium-mediated plant transformation

    PubMed Central

    Anand, Ajith; Mysore, Kirankumar S

    2013-01-01

    RAR1 is identified as a critical protein involved in plant innate immunity. We investigated the role of RAR1 in Agrobacterium-mediated plant transformation based on the previous findings that accessory proteins associated with the E3 ligase complex such as SGT1, which tightly interacts with RAR1, play a role in the transformation process. RAR1 gene silencing in Nicotiana benthamiana and Arabidopsis rar1 mutant analysis suggested that RAR1 is required for early stages of Agrobacterium-mediated plant transformation. This finding further illustrates that RAR1, along with SGT1, that serve as a HSP90 co-chaperone is important for Agrobacterium-mediated plant transformation.

  6. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori.

    PubMed

    Michielse, C B; Ram, A F J; Hooykaas, P J J; Hondel, C A M J J van den

    2004-05-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important. PMID:15050546

  7. Genetic control and regulatory mechanisms of succinoglycan and curdlan biosynthesis in genus Agrobacterium.

    PubMed

    Wu, Dan; Li, Ang; Ma, Fang; Yang, Jixian; Xie, Yutong

    2016-07-01

    Agrobacterium is a genus of gram-negative bacteria that can produce several typical exopolysaccharides with commercial uses in the food and pharmaceutical fields. In particular, succinoglycan and curdlan, due to their good quality in high yield, have been employed on an industrial scale comparatively early. Exopolysaccharide biosynthesis is a multiple-step process controlled by different functional genes, and various environmental factors cause changes in exopolysaccharide biosynthesis through regulatory mechanisms. In this mini-review, we focus on the genetic control and regulatory mechanisms of succinoglycan and curdlan produced by Agrobacterium. Some key functional genes and regulatory mechanisms for exopolysaccharide biosynthesis are described, possessing a high potential for application in metabolic engineering to modify exopolysaccharide production and physicochemical properties. This review may contribute to the understanding of exopolysaccharide biosynthesis and exopolysaccharide modification by metabolic engineering methods in Agrobacterium. PMID:27255488

  8. A high-efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.).

    PubMed

    Ozawa, Kenjirou

    2012-01-01

    Agrobacterium-mediated transformation of rice has been routinely performed according to the protocol reported by Hiei et al. (Plant J. 6:271-282, 1994). However, several elite japonica and many indica varieties cannot be efficiently transformed by Agrobacterium system. Also a large number of transformants are required to generate T-DNA insertion and FOX libraries as well as gene-targeting studies. To overcome these challenges, we established a high-efficiency transformation system in rice by cocultivating rice calli with Agrobacterium on filter papers moistened with enriched liquid media instead of using solid media (Ozawa, Plant Sci. 176:522-527, 2009; Ozawa and Takaiwa, Plant Sci. 179:333-337, 2010). In this system, the transformation efficiency of the calli is almost 100% in many varieties.

  9. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582

    NASA Astrophysics Data System (ADS)

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S.; Ellis, Tom

    2016-03-01

    Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.

  10. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

    PubMed

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S; Ellis, Tom

    2016-01-01

    Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity. PMID:27010592

  11. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582

    PubMed Central

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S.; Ellis, Tom

    2016-01-01

    Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity. PMID:27010592

  12. Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582.

    PubMed

    Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S; Ellis, Tom

    2016-03-24

    Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.

  13. Advances in Agrobacterium tumefaciens-mediated genetic transformation of graminaceous crops.

    PubMed

    Singh, Roshan Kumar; Prasad, Manoj

    2016-05-01

    Steady increase in global population poses several challenges to plant science research, including demand for increased crop productivity, grain yield, nutritional quality and improved tolerance to different environmental factors. Transgene-based approaches are promising to address these challenges by transferring potential candidate genes to host organisms through different strategies. Agrobacterium-mediated gene transfer is one such strategy which is well known for enabling efficient gene transfer in both monocot and dicots. Due to its versatility, this technique underwent several advancements including development of improved in vitro plant regeneration system, co-cultivation and selection methods, and use of hyper-virulent strains of Agrobacterium tumefaciens harbouring super-binary vectors. The efficiency of this method has also been enhanced by the use of acetosyringone to induce the activity of vir genes, silver nitrate to reduce the Agrobacterium-induced necrosis and cysteine to avoid callus browning during co-cultivation. In the last two decades, extensive efforts have been invested towards achieving efficient Agrobacterium-mediated transformation in cereals. Though high-efficiency transformation systems have been developed for rice and maize, comparatively lesser progress has been reported in other graminaceous crops. In this context, the present review discusses the progress made in Agrobacterium-mediated transformation system in rice, maize, wheat, barley, sorghum, sugarcane, Brachypodium, millets, bioenergy and forage and turf grasses. In addition, it also provides an overview of the genes that have been recently transferred to these graminaceous crops using Agrobacterium, bottlenecks in this technique and future possibilities for crop improvement. PMID:26660352

  14. Complete Genome Sequence of the Larval Shellfish Pathogen Vibrio tubiashii Type Strain ATCC 19109.

    PubMed

    Richards, Gary P; Needleman, David S; Watson, Michael A; Bono, James L

    2014-12-18

    Vibrio tubiashii is a larval shellfish pathogen. Here, we report the first closed genome sequence for this species (ATCC type strain 19109), which consists of two chromosomes (3,294,490 and 1,766,582 bp), two megaplasmids (251,408 and 122,808 bp), and two plasmids (57,076 and 47,973 bp).

  15. Complete Genome Sequence of Thiostrepton-Producing Streptomyces laurentii ATCC 31255

    PubMed Central

    Fujino, Yasuhiro; Nagayoshi, Yuko; Ohshima, Toshihisa; Ogata, Seiya

    2016-01-01

    Streptomyces laurentii ATCC 31255 produces thiostrepton, a thiopeptide class antibiotic. Here, we report the complete genome sequence for this strain, which contains a total of 8,032,664 bp, 7,452 predicted coding sequences, and a G+C content of 72.3%. PMID:27257211

  16. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

    PubMed Central

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-01-01

    Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain. PMID:27305971

  17. Complete Genome Sequence of Brachyspira hyodysenteriae Type Strain B-78 (ATCC 27164).

    PubMed

    Mirajkar, Nandita S; Johnson, Timothy J; Gebhart, Connie J

    2016-01-01

    Reported herein is the complete genome sequence of the type strain B-78 (ATCC 27164) of Brachyspira hyodysenteriae, the etiological agent of swine dysentery. The 3.1-Mb genome consists of a 3.056-Mb chromosome and a 45-kb plasmid, with 2,617 protein-coding genes, 39 RNA genes, and 40 pseudogenes. PMID:27540064

  18. Finished Genome Sequence of the Laboratory Strain Escherichia coli K-12 RV308 (ATCC 31608).

    PubMed

    Krempl, Peter M; Mairhofer, Juergen; Striedner, Gerald; Thallinger, Gerhard G

    2014-11-20

    Escherichia coli strain K-12 substrain RV308 is an engineered descendant of the K-12 wild-type strain. Like its ancestor, it is an important organism in biotechnological research and is heavily used for the expression of single-chain variable fragments. Here, we report the complete genome sequence of E. coli K-12 RV308 (ATCC 31608).

  19. Finished Genome Sequence of Escherichia coli K-12 Strain HMS174 (ATCC 47011).

    PubMed

    Mairhofer, Juergen; Krempl, Peter M; Thallinger, Gerhard G; Striedner, Gerald

    2014-11-20

    Escherichia coli strain K-12 substrain HMS174 is an engineered descendant of the E. coli K-12 wild-type strain. Like its ancestor, it is an important organism in biotechnological research and is used in fermentation processes for heterologous protein production. Here, we report the complete genome sequence of E. coli HMS174 (ATCC 47011).

  20. Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock

    PubMed Central

    Chang, Philip Lee; Yen, Teh Fu

    1985-01-01

    Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed. PMID:16346956

  1. Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock.

    PubMed

    Chang, P L; Yen, T F

    1985-12-01

    Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed.

  2. Properties of a hydrogen-inhibited mutant of Desulfovibrio desulfuricans ATCC 27774.

    PubMed Central

    Odom, J M; Wall, J D

    1987-01-01

    A mutant of Desulfovibrio desulfuricans ATCC 27774 has been obtained which is incapable of sulfate respiration with molecular hydrogen but which grows normally on lactate plus sulfate under argon. Growth characteristics of the mutant suggest that the defect is involved in electron transfer to sulfate or nitrate but not thiosulfate. PMID:3818548

  3. Complete Genome Sequence of Thiostrepton-Producing Streptomyces laurentii ATCC 31255.

    PubMed

    Doi, Katsumi; Fujino, Yasuhiro; Nagayoshi, Yuko; Ohshima, Toshihisa; Ogata, Seiya

    2016-06-02

    Streptomyces laurentii ATCC 31255 produces thiostrepton, a thiopeptide class antibiotic. Here, we report the complete genome sequence for this strain, which contains a total of 8,032,664 bp, 7,452 predicted coding sequences, and a G+C content of 72.3%.

  4. Complete Genome Sequence and Methylome Analysis of Bacillus globigii ATCC 49760.

    PubMed

    Morgan, Richard D

    2016-05-26

    Bacillus subtilis (Ehrenburg) Cohn ATCC 49760, deposited as Bacillus globigii, is the source strain for the restriction enzymes BglI and BglII. Its complete sequence and full methylome were determined using single-molecule real-time (SMRT) sequencing.

  5. Genome Sequence of Streptomyces viridosporus Strain T7A ATCC 39115, a Lignin-Degrading Actinomycete

    SciTech Connect

    Davis, Jennifer R.; Goodwin, Lynne A.; Teshima, Hazuki; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Huntemann, Marcel; Wei, Chia-Lin; Han, James; Chen, Amy; Kyrpides, Nikos C; Mavromatis, K; Szeto, Ernest; Markowitz, Victor; Ivanova, N; Mikhailova, Natalia; Ovchinnikova, Galina; Pagani, Ioanna; Pati, Amrita; Woyke, Tanja; Pitluck, Sam; Peters, Lin; Nolan, Matt; Land, Miriam L; Sello, Jason K.

    2013-01-01

    We announce the availability of the genome sequence of Streptomyces viridosporus strain T7A ATCC 39115, a plant biomass- degrading actinomycete. This bacterium is of special interest because of its capacity to degrade lignin, an underutilized compo- nent of plants in the context of bioenergy. It has a full complement of genes for plant biomass catabolism.

  6. Draft Genome Assembly of Bordetella bronchiseptica ATCC 10580, a Historical Canine Clinical Isolate.

    PubMed

    Daligault, H E; Davenport, K W; Minogue, T D; Bishop-Lilly, K A; Bruce, D C; Chain, P S; Coyne, S R; Frey, K G; Jaissle, J; Koroleva, G I; Ladner, J T; Lo, C-C; Meincke, L; Munk, C; Palacios, G F; Redden, C L; Johnson, S L

    2014-01-01

    We present the scaffolded genome of Bordetella bronchiseptica ATCC 10580, assembled into 98 contigs. This 5.1-Mb assembly (68.2% G+C content) contains 4,870 coding regions. The strain was originally isolated from canine lung tissue and is used in quality control testing. PMID:25237025

  7. Genome Assembly of Shigella flexneri ATCC 12022, a Quality Control Reference Strain

    PubMed Central

    Daligault, H. E.; Davenport, K. W.; Minogue, T. D.; Bishop-Lilly, K. A.; Broomall, S. M.; Bruce, D. C.; Chain, P. S.; Coyne, S. R.; Freitas, T.; Frey, K. G.; Gibbons, H. S.; Jaissle, J.; Lo, C.-C.; Meincke, L.; Munk, A. C.; Redden, C. L.; Rosenzweig, C. N.

    2014-01-01

    Shigella flexneri causes shigellosis, severe and potentially life-threatening diarrhea, and accounts for 18% of shigellosis cases in the United States. Here, we present the 4.51-Mbp genome assembly of S. flexneri ATCC 12022, a quality control and reference strain, in 10 scaffolds. PMID:25359907

  8. Complete Genome Sequence of the Quality Control Strain Staphylococcus aureus subsp. aureus ATCC 25923

    PubMed Central

    Treangen, Todd J.; Maybank, Rosslyn A.; Enke, Sana; Friss, Mary Beth; Diviak, Lynn F.; Karaolis, David K. R.; Koren, Sergey; Ondov, Brian; Phillippy, Adam M.; Bergman, Nicholas H.

    2014-01-01

    Staphylococcus aureus subsp. aureus ATCC 25923 is commonly used as a control strain for susceptibility testing to antibiotics and as a quality control strain for commercial products. We present the completed genome sequence for the strain, consisting of the chromosome and a 27.5-kb plasmid. PMID:25377701

  9. Complete Genome Sequence of the Quality Control Strain Staphylococcus aureus subsp. aureus ATCC 25923.

    PubMed

    Treangen, Todd J; Maybank, Rosslyn A; Enke, Sana; Friss, Mary Beth; Diviak, Lynn F; Karaolis, David K R; Koren, Sergey; Ondov, Brian; Phillippy, Adam M; Bergman, Nicholas H; Rosovitz, M J

    2014-01-01

    Staphylococcus aureus subsp. aureus ATCC 25923 is commonly used as a control strain for susceptibility testing to antibiotics and as a quality control strain for commercial products. We present the completed genome sequence for the strain, consisting of the chromosome and a 27.5-kb plasmid. PMID:25377701

  10. Complete Genome Sequence of Brachyspira hyodysenteriae Type Strain B-78 (ATCC 27164)

    PubMed Central

    Mirajkar, Nandita S.; Johnson, Timothy J.

    2016-01-01

    Reported herein is the complete genome sequence of the type strain B-78 (ATCC 27164) of Brachyspira hyodysenteriae, the etiological agent of swine dysentery. The 3.1-Mb genome consists of a 3.056-Mb chromosome and a 45-kb plasmid, with 2,617 protein-coding genes, 39 RNA genes, and 40 pseudogenes. PMID:27540064

  11. Effect of Calcium in Assay Medium on D Value of Bacillus stearothermophilus ATCC 7953 Spores

    PubMed Central

    Sasaki, Koichi; Shintani, Hideharu; Itoh, Junpei; Kamogawa, Takuji; Kajihara, Yousei

    2000-01-01

    The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of D value. PMID:11097939

  12. Draft Genome Sequence of the Probiotic Strain Lactobacillus acidophilus ATCC 4356

    PubMed Central

    Palomino, Maria Mercedes; Allievi, Mariana C.; Fina Martin, Joaquina; Waehner, Pablo M.; Prado Acosta, Mariano; Sanchez Rivas, Carmen

    2015-01-01

    We present the 1,956,699-bp draft genome sequence of Lactobacillus acidophilus strain ATCC 4356. Comparative genomic analysis revealed 99.96% similarity with L. acidophilus NCFM NC_006814.3 and 99.97% with La-14 NC_021181.2 genomes. PMID:25593259

  13. Antimicrobial mechanism of flavonoids against Escherichia coli ATCC 25922 by model membrane study

    NASA Astrophysics Data System (ADS)

    He, Mengying; Wu, Ting; Pan, Siyi; Xu, Xiaoyun

    2014-06-01

    Antimicrobial mechanism of four flavonoids (kaempferol, hesperitin, (+)-catechin hydrate, biochanin A) against Escherichia coli ATCC 25922 was investigated through cell membranes and a liposome model. The release of bacterial protein and images from transmission electron microscopy demonstrated damage to the E. coli ATCC 25922 membrane. A liposome model with dipalmitoylphosphatidylethanolamine (DPPE) (0.6 molar ratio) and dipalmitoylphosphatidylglycerol (DPPG) (0.4 molar ratio), representative of the phospholipid membrane of E. coli ATCC 25922, was used to specify the mode of action of four selected flavonoids through Raman spectroscopy and differential scanning calorimetry. It is suggested that for flavonoids, to be effective antimicrobials, interaction with the polar head-group of the model membrane followed by penetration into the hydrophobic regions must occur. The antimicrobial efficacies of the flavonoids were consistent with liposome interaction activities, kaempferol > hesperitin > (+)-catechin hydrate > biochanin A. This study provides a liposome model capable of mimicking the cell membrane of E. coli ATCC 25922. The findings are important in understanding the antibacterial mechanism on cell membranes.

  14. Complete genome sequence of the beer spoilage organism Pediococcus claussenii ATCC BAA-344T.

    PubMed

    Pittet, Vanessa; Abegunde, Teju; Marfleet, Travis; Haakensen, Monique; Morrow, Kendra; Jayaprakash, Teenus; Schroeder, Kristen; Trost, Brett; Byrns, Sydney; Bergsveinson, Jordyn; Kusalik, Anthony; Ziola, Barry

    2012-03-01

    Pediococcus claussenii is a common brewery contaminant. We have sequenced the chromosome and plasmids of the type strain P. claussenii ATCC BAA-344. A ropy variant was chosen for sequencing to obtain genetic information related to growth in beer, as well as exopolysaccharide and possibly biofilm formation by this organism. PMID:22328764

  15. Draft Genome Sequence of Streptomyces silvensis ATCC 53525, a Producer of Novel Hormone Antagonists

    PubMed Central

    Johnston, Chad W.; Li, Yongchang

    2016-01-01

    Streptomyces silvensis produces nonribosomal peptides that act as antagonists of the human oxytocin and vasopressin receptors. Here, we present the genome sequence of S. silvensis ATCC 53525 and demonstrate that this organism possesses a number of additional biosynthetic gene clusters and might be a promising source for genome-guided drug discovery efforts. PMID:26893408

  16. Draft Genome Assembly of Bordetella bronchiseptica ATCC 10580, a Historical Canine Clinical Isolate.

    PubMed

    Daligault, H E; Davenport, K W; Minogue, T D; Bishop-Lilly, K A; Bruce, D C; Chain, P S; Coyne, S R; Frey, K G; Jaissle, J; Koroleva, G I; Ladner, J T; Lo, C-C; Meincke, L; Munk, C; Palacios, G F; Redden, C L; Johnson, S L

    2014-01-01

    We present the scaffolded genome of Bordetella bronchiseptica ATCC 10580, assembled into 98 contigs. This 5.1-Mb assembly (68.2% G+C content) contains 4,870 coding regions. The strain was originally isolated from canine lung tissue and is used in quality control testing.

  17. Ecological dynamics and complex interactions of Agrobacterium megaplasmids.

    PubMed

    Platt, Thomas G; Morton, Elise R; Barton, Ian S; Bever, James D; Fuqua, Clay

    2014-01-01

    As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it's Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together. PMID:25452760

  18. Binding-protein-dependent lactose transport in Agrobacterium radiobacter.

    PubMed

    Greenwood, J A; Cornish, A; Jones, C W

    1990-04-01

    Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.

  19. Ecological dynamics and complex interactions of Agrobacterium megaplasmids

    PubMed Central

    Platt, Thomas G.; Morton, Elise R.; Barton, Ian S.; Bever, James D.; Fuqua, Clay

    2014-01-01

    As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it’s Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together. PMID:25452760

  20. Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579.

    PubMed

    Tagawa, Yuichi

    2014-12-01

    Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.

  1. Identification of Campylobacter jejuni ATCC 43431-Specific Genes by Whole Microbial Genome Comparisons

    PubMed Central

    Poly, Frédéric; Threadgill, Deborah; Stintzi, Alain

    2004-01-01

    This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis. PMID:15231810

  2. Processing of cellulosic material by a cellulase-containing cell-free fermentate produced from cellulase-producing bacteria, ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-08-04

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate, have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase degrading bacterium ATCC 55702, which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic materials. 5 figs.

  3. Processing of cellulosic material by a cellulase-containing cell-free fermentate produced from cellulase-producing bacteria, ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate, have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase degrading bacterium ATCC 55702, which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic materials.

  4. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

    PubMed

    Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

    2016-04-01

    The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples. PMID:26593465

  5. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

    PubMed

    Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

    2016-04-01

    The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples.

  6. [Induction and in vitro culture of hairy roots of Dianthus caryophyllus and its plant regeneration].

    PubMed

    Shi, Heping; Zhu, Yuanfeng; Wang, Bei; Sun, Jiangbing; Huang, Shengqin

    2014-11-01

    To use Agrobacterium rhizogenes-induced hairy roots to create new germplasm of Dianthus caryophyllus, we transformed D. caryophyllus with A. rhizogenes by leaf disc for plant regeneration from hairy roots. The white hairy roots could be induced from the basal surface of leaf explants of D. caryophyllus 12 days after inoculation with A. rhizogenes ATCC15834. The percentage of the rooting leaf explants was about 90% 21 days after inoculation. The hairy roots could grow rapidly and autonomously in liquid or solid phytohormone-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and silica gel thin-layer chromatography of opines from D. caryophyllus hairy roots. Hairy roots could form light green callus after cultured on MS+6-BA 1.0-3.0 mg/L + NAA 0.1-0.2 mg/L for 15 days. The optimum medium for adventitious shoots formation was MS + 6-BA 2.0 mg/L + NAA 0.02 mg/L, where the rate of adventitious shoot induction was 100% after cultured for 6 weeks. The mean number of adventitious shoot per callus was 30-40. The adventitious shoots can form roots when cultured on phytohormone-free 1/2 MS or 1/2 MS +0.5 mg/L NAA for 10 days. When the rooted plantlets transplanted in the substrate mixed with perlite sand and peat (volume ratio of 1:2), the survival rate was above 95%.

  7. Lactobacillus acidophilus ATCC 4356 prevents atherosclerosis via inhibition of intestinal cholesterol absorption in apolipoprotein E-knockout mice.

    PubMed

    Huang, Ying; Wang, Jinfeng; Quan, Guihua; Wang, Xiaojun; Yang, Longfei; Zhong, Lili

    2014-12-01

    The objective of this study was to investigate the effect of Lactobacillus acidophilus ATCC 4356 on the development of atherosclerosis in apolipoprotein E-knockout (ApoE(-/-)) mice. Eight-week-old ApoE(-/-) mice were fed a Western diet with or without L. acidophilus ATCC 4356 daily for 16 weeks. L. acidophilus ATCC 4356 protected ApoE(-/-) mice from atherosclerosis by reducing their plasma cholesterol levels from 923 ± 44 to 581 ± 18 mg/dl, likely via a marked decrease in cholesterol absorption caused by modulation of Niemann-Pick C1-like 1 (NPC1L1). In addition, suppression of cholesterol absorption induced reverse cholesterol transport (RCT) in macrophages through the peroxisome proliferator-activated receptor/liver X receptor (PPAR/LXR) pathway. Fecal lactobacillus and bifidobacterium counts were significantly (P < 0.05) higher in the L. acidophilus ATCC 4356 treatment groups than in the control groups. Furthermore, L. acidophilus ATCC 4356 was detected in the rat small intestine, colon, and feces during the feeding trial. The bacterial levels remained high even after the administration of lactic acid bacteria had been stopped for 2 weeks. These results suggest that administration of L. acidophilus ATCC 4356 can protect against atherosclerosis through the inhibition of intestinal cholesterol absorption. Therefore, L. acidophilus ATCC 4356 may be a potential therapeutic material for preventing the progression of atherosclerosis.

  8. Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens.

    PubMed

    Anand, Ajith; Uppalapati, Srinivasa Rao; Ryu, Choong-Min; Allen, Stacy N; Kang, Li; Tang, Yuhong; Mysore, Kirankumar S

    2008-02-01

    We investigated the effects of salicylic acid (SA) and systemic acquired resistance (SAR) on crown gall disease caused by Agrobacterium tumefaciens. Nicotiana benthamiana plants treated with SA showed decreased susceptibility to Agrobacterium infection. Exogenous application of SA to Agrobacterium cultures decreased its growth, virulence, and attachment to plant cells. Using Agrobacterium whole-genome microarrays, we characterized the direct effects of SA on bacterial gene expression and showed that SA inhibits induction of virulence (vir) genes and the repABC operon, and differentially regulates the expression of many other sets of genes. Using virus-induced gene silencing, we further demonstrate that plant genes involved in SA biosynthesis and signaling are important determinants for Agrobacterium infectivity on plants. Silencing of ICS (isochorismate synthase), NPR1 (nonexpresser of pathogenesis-related gene 1), and SABP2 (SA-binding protein 2) in N. benthamiana enhanced Agrobacterium infection. Moreover, plants treated with benzo-(1,2,3)-thiadiazole-7-carbothioic acid, a potent inducer of SAR, showed reduced disease symptoms. Our data suggest that SA and SAR both play a major role in retarding Agrobacterium infectivity. PMID:18156296

  9. Stability of free and encapsulated Lactobacillus acidophilus ATCC 4356 in yogurt and in an artificial human gastric digestion system.

    PubMed

    Ortakci, F; Sert, S

    2012-12-01

    The objective of this study was to determine the effect of encapsulation on survival of probiotic Lactobacillus acidophilus ATCC 4356 (ATCC 4356) in yogurt and during artificial gastric digestion. Strain ATCC 4356 was added to yogurt either encapsulated in calcium alginate or in free form (unencapsulated) at levels of 8.26 and 9.47 log cfu/g, respectively, and the influence of alginate capsules (1.5 to 2.5mm) on the sensorial characteristics of yogurts was investigated. The ATCC 4356 strain was introduced into an artificial gastric solution consisting of 0.08 N HCl (pH 1.5) containing 0.2% NaCl or into artificial bile juice consisting of 1.2% bile salts in de Man, Rogosa, and Sharpe broth to determine the stability of the probiotic bacteria. When incubated for 2h in artificial gastric juice, the free ATCC 4356 did not survive (reduction of >7 log cfu/g). We observed, however, greater survival of encapsulated ATCC 4356, with a reduction of only 3 log cfu/g. Incubation in artificial bile juice (6 h) did not significantly affect the viability of free or encapsulated ATCC 4356. Moreover, statistically significant reductions (~1 log cfu/g) of both free and encapsulated ATCC 4356 were observed during 4-wk refrigerated storage of yogurts. The addition of probiotic cultures in free or alginate-encapsulated form did not significantly affect appearance/color or flavor/odor of the yogurts. However, significant deficiencies were found in body/texture of yogurts containing encapsulated ATCC 4356. We concluded that incorporation of free and encapsulated probiotic bacteria did not substantially change the overall sensory properties of yogurts, and encapsulation in alginate using the extrusion method greatly enhanced the survival of probiotic bacteria against an artificial human gastric digestive system.

  10. Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation.

    PubMed

    Kwon, Tackmin

    2016-09-01

    The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection. PMID:27643450

  11. A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated genetic transformation.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2013-01-01

    Agrobacterium is a phytopathogenic bacterium that induces crown gall disease in many plant species by transferring and integrating a segment of its own DNA (T-DNA) into its host genome. Whereas Agrobacterium usually does not trigger an extensive defense response in its host plants, it induces the expression of several defense-related genes and activates plant stress reactions. In the complex interplay between Agrobacterium and its host plant, Agrobacterium has evolved to take advantage of these plant defense pathways for its own purpose of advancement of the infection process. For example, Agrobacterium utilizes the host stress response transcriptional regulator VIP1 to facilitate nuclear import and proteasomal uncoating of its T-DNA during genetic transformation of the host cell. In Arabidopsis, the VIP1 gene expression is repressed by WRKY17, a negative regulator of basal resistance to Pseudomonas. Thus, we examined whether WRKY17 is also involved in plant susceptibility to genetic transformation by Agrobacterium. Using reverse genetics, we showed that a wrky17 mutant displays higher expression of the VIP1 gene in roots, but not in shoots. In a root infection assay, the wrky17 mutant plants were hyper-susceptible to Agrobacterium compared to wild type plants. WRKY17, therefore, may act as a positive regulator of Arabidopsis resistance to Agrobacterium. This notion is important for understanding the complex regulation of Agrobacterium-mediated genetic transformation; thus, although this paper reports a relatively small set of data that we do not plan to pursue further in our lab, we believe it might be useful for the broad community of plant pathologists and plant biotechnologists. PMID:24358874

  12. Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation

    PubMed Central

    Kwon, Tackmin

    2016-01-01

    The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection. PMID:27643450

  13. A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated genetic transformation

    PubMed Central

    Lacroix, Benoît; Citovsky, Vitaly

    2013-01-01

    Agrobacterium is a phytopathogenic bacterium that induces crown gall disease in many plant species by transferring and integrating a segment of its own DNA (T-DNA) into its host genome. Whereas Agrobacterium usually does not trigger an extensive defense response in its host plants, it induces the expression of several defense-related genes and activates plant stress reactions. In the complex interplay between Agrobacterium and its host plant, Agrobacterium has evolved to take advantage of these plant defense pathways for its own purpose of advancement of the infection process. For example, Agrobacterium utilizes the host stress response transcriptional regulator VIP1 to facilitate nuclear import and proteasomal uncoating of its T-DNA during genetic transformation of the host cell. In Arabidopsis, the VIP1 gene expression is repressed by WRKY17, a negative regulator of basal resistance to Pseudomonas. Thus, we examined whether WRKY17 is also involved in plant susceptibility to genetic transformation by Agrobacterium. Using reverse genetics, we showed that a wrky17 mutant displays higher expression of the VIP1 gene in roots, but not in shoots. In a root infection assay, the wrky17 mutant plants were hyper-susceptible to Agrobacterium compared to wild type plants. WRKY17, therefore, may act as a positive regulator of Arabidopsis resistance to Agrobacterium. This notion is important for understanding the complex regulation of Agrobacterium-mediated genetic transformation; thus, although this paper reports a relatively small set of data that we do not plan to pursue further in our lab, we believe it might be useful for the broad community of plant pathologists and plant biotechnologists. PMID:24358874

  14. Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera.

    PubMed

    Maheshwari, Priti; Kovalchuk, Igor

    2016-01-01

    The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar - Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development. PMID:27014319

  15. Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process

    PubMed Central

    Jia, Yuying; Yao, Xingdong; Zhao, Mingzhe; Zhao, Qiang; Du, Yanli; Yu, Cuimei; Xie, Futi

    2015-01-01

    The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively. PMID:26262617

  16. DETECTION AND IMPLICATIONS OF EARLY AGROBACTERIUM TUMEFACIENS INFECTION OF PARADOX SEEDS AND SEEDLINGS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paradox (Juglans hindsii x J. regia), the dominant rootstock used in California, USA walnut production, has many desirable horticultural characteristics, but is highly susceptible to crown gall. Crown gall, caused by the soil-borne bacterium Agrobacterium tumefaciens, can not be consistently control...

  17. Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera

    PubMed Central

    Maheshwari, Priti; Kovalchuk, Igor

    2016-01-01

    The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar – Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development. PMID:27014319

  18. Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera.

    PubMed

    Maheshwari, Priti; Kovalchuk, Igor

    2016-01-01

    The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar - Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development.

  19. Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production.

    PubMed

    Jones, Richard W

    2016-01-01

    When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced. PMID:26658852

  20. Novel primers for detection of genetically diverse virulent Agrobacterium tumefaciens bv1 strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel primers were developed to amplify a 243 bp fragment of an intergenic region between gene5 and tms2 on the T-DNA of Agrobacterium tumefaciens. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence and did not generate extraneous bands,...

  1. X-ray Structure of Imidazolonepropionase from Agrobacterium tumefaciens at 1.87 angstrom Resolution

    SciTech Connect

    Tyagi,R.; Kumaran, D.; Burley, S.; Swaminathan, S.

    2007-01-01

    Histidine degradation in agrobacterium tumefaciens involves four enzymes, including histidase, urocanase, imidazolonepropionase, and N-formylglutamate amido hydrolase. The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-t-glutamate.

  2. Agroinfiltration by cytokinin-producing Agrobacterium sp. strain GV3101 primes defense responses in Nicotiana tabacum.

    PubMed

    Sheikh, Arsheed Hussain; Raghuram, Badmi; Eschen-Lippold, Lennart; Scheel, Dierk; Lee, Justin; Sinha, Alok Krishna

    2014-11-01

    Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies. PMID:25054409

  3. Agrobacterium tumefaciens-mediated transformation of corn (Zea mays L.) multiple shoots

    PubMed Central

    Cao, Shi-liang; Masilamany, Pathmalojiny; Li, Wen-bin; Pauls, K. Peter

    2014-01-01

    An Agrobacterium tumefaciens-mediated corn transformation method based on multiple shoot tissue cultures was developed, which is effective with a variety of corn inbred lines and standard binary vectors. Six factors that affected the success of corn transformation were tested, including A. tumefaciens strain, corn genotype, tissue culture growth stage, medium composition, co-culture temperature and surfactant treatment. Agropine-type bacteria (EHA 101 and AGL 1) were eightfold more effective than octopine-type strain for corn multi-shoot tissues transformation. The average frequency of Glucuronidase (GUS)-positive explants obtained from 14 corn genotypes ranged from 36% to 76%. L-proline (0.7 g L−1) in the co-culture medium apparently improved the frequency of transformation. The newly initiated multi-shoot tissues were most responsive to Agrobacterium infection. A positive correlation was found between multi-shoot tissue susceptibility to Agrobacterium and the proportion of cells in G1 phase. Transformants were identified by reverse transcription Polymerase Chain Reaction (PCR) and by southern blot hybridization assays. The frequency of transformants was approximately 2% based on the number of multi-shoot explants co-cultivated with Agrobacterium. PMID:26019506

  4. Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process.

    PubMed

    Jia, Yuying; Yao, Xingdong; Zhao, Mingzhe; Zhao, Qiang; Du, Yanli; Yu, Cuimei; Xie, Futi

    2015-08-07

    The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

  5. Case of bacterial endophthalmitis caused by an Agrobacterium radiobacter-like organism.

    PubMed Central

    Miller, J M; Novy, C; Hiott, M

    1996-01-01

    A case of postsurgical endophthalmitis caused by Agrobacterium radiobacter in a 70-year-old male is reported. A. radiobacter organisms are normally environmental bacteria but may occasionally be opportunistic pathogens. Infection in this case occurred after the patient was discharged following routine cataract surgery. The infection cleared after empiric therapy intraocular administration of vancomycin and gentamicin. PMID:8940475

  6. Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).

    PubMed

    Mhatre, Minal

    2013-01-01

    Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern.

  7. Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).

    PubMed

    Mhatre, Minal

    2013-01-01

    Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern. PMID:23179718

  8. Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha using regenerating thalli.

    PubMed

    Kubota, Akane; Ishizaki, Kimitsune; Hosaka, Masashi; Kohchi, Takayuki

    2013-01-01

    The thallus, the gametophyte body of the liverwort Marchantia polymorpha, develops clonal progenies called gemmae that are useful in the isolation and propagation of isogenic plants. Developmental timing is critical to Agrobacterium-mediated transformation, and high transformation efficiency has been achieved only with sporelings. Here we report an Agrobacterium-mediated transformation system for M. polymorpha using regenerating thalli. Thallus regeneration was induced by cutting the mature thallus across the apical-basal axis and incubating the basal portion of the thallus for 3 d. Regenerating thalli were infected with Agrobacterium carrying binary vector that contained a selection marker, the hygromycin phosphotransferase gene, and hygromycin-resistant transformants were obtained with an efficiency of over 60%. Southern blot analysis verified random integration of 1 to 4 copies of the T-DNA into the M. polymorpha genome. This Agrobacterium-mediated transformation system for M. polymorpha should provide opportunities to perform genetic transformation without preparing spores and to generate a sufficient number of transformants with isogenic background. PMID:23291762

  9. Agrobacterium-mediated transformation of the recalcitrant Vanda Kasem's Delight orchid with higher efficiency.

    PubMed

    Gnasekaran, Pavallekoodi; Antony, Jessica Jeyanthi James; Uddain, Jasim; Subramaniam, Sreeramanan

    2014-01-01

    The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A 600 nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 μM acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

  10. Agrobacterium-mediated transformation of the white-rot fungus Physisporinus vitreus.

    PubMed

    Schubert, M; Stührk, C; Fuhr, M J; Schwarze, F W M R

    2013-11-01

    The biotechnologically important white-rot fungus Physisporinus vitreus was co-cultivated with Agrobacterium tumefaciens AGL-1 carrying plasmids with nourseothricin resistance as the selectable marker gene and red fluorescence protein as a visual marker. Mitotically stable transformed isolates were obtained showing red fluorescence protein activity.

  11. An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars.

    PubMed

    Shri, Manju; Rai, Arti; Verma, Pankaj Kumar; Misra, Prashant; Dubey, Sonali; Kumar, Smita; Verma, Sikha; Gautam, Neelam; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

    2013-04-01

    Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of L-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD600 nm = 0.5-0.8) promoted the highest frequency of transformation (83.04 %) in medium containing L-cysteine (400 mg l(-1)). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of L-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.

  12. Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration.

    PubMed

    Bakshi, Souvika; Sadhukhan, Ayan; Mishra, Sagarika; Sahoo, Lingaraj

    2011-12-01

    An improved method of Agrobacterium-mediated transformation of cowpea was developed employing both sonication and vacuum infiltration treatments. 4 day-old cotyledonary nodes were used as explants for co-cultivation with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pSouv-cry1Ac. Among the different injury treatments, vacuum infiltration and their combination treatments tested, sonication for 20 s followed by vacuum infiltration for 5 min with A. tumefaciens resulted in highest transient GUS expression efficiency (93% explants expressing GUS at regenerating sites). After 3 days of co-cultivation, the explants were cultured in 150 mg/l kanamycin-containing selection medium and putative transformed plants were recovered. The presence, integration and expression of nptII and cry1Ac genes in T0 transgenic plants were confirmed by polymerase chain reaction (PCR), genomic Southern and qualitative reverse transcription (RT)-PCR analysis. Western blot hybridization and enzyme-linked immunosorbent assay (ELISA) detected and demonstrated the accumulation of Cry1Ac protein in transgenic plants. The cry1Ac gene transmitted in a Mendelian fashion. The stable transformation efficiency increased by 88.4% using both sonication-assisted Agrobacterium-mediated transformation (SAAT) and vacuum infiltration than simple Agrobacterium-mediated transformation in cowpea.

  13. Agrobacterium-Mediated Transformation of Bread and Durum Wheat Using Freshly Isolated Immature Embryos

    NASA Astrophysics Data System (ADS)

    Huixia, Wu; Angela, Doherty; Jones, Huw D.

    Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.

  14. Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha using regenerating thalli.

    PubMed

    Kubota, Akane; Ishizaki, Kimitsune; Hosaka, Masashi; Kohchi, Takayuki

    2013-01-01

    The thallus, the gametophyte body of the liverwort Marchantia polymorpha, develops clonal progenies called gemmae that are useful in the isolation and propagation of isogenic plants. Developmental timing is critical to Agrobacterium-mediated transformation, and high transformation efficiency has been achieved only with sporelings. Here we report an Agrobacterium-mediated transformation system for M. polymorpha using regenerating thalli. Thallus regeneration was induced by cutting the mature thallus across the apical-basal axis and incubating the basal portion of the thallus for 3 d. Regenerating thalli were infected with Agrobacterium carrying binary vector that contained a selection marker, the hygromycin phosphotransferase gene, and hygromycin-resistant transformants were obtained with an efficiency of over 60%. Southern blot analysis verified random integration of 1 to 4 copies of the T-DNA into the M. polymorpha genome. This Agrobacterium-mediated transformation system for M. polymorpha should provide opportunities to perform genetic transformation without preparing spores and to generate a sufficient number of transformants with isogenic background.

  15. Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.

    PubMed

    Bull, S E; Owiti, J A; Niklaus, M; Beeching, J R; Gruissem, W; Vanderschuren, H

    2009-01-01

    Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.

  16. Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.).

    PubMed

    Ozawa, Kenjirou

    2009-04-01

    Technologies for transformation of rice have been developed to meet the requirements of functional genomics in order to enable the production of transgenic rice plants with useful agricultural characters. However, many rice varieties are not efficiently transformed by Agrobacterium. We have succeeded in establishing a highly efficient transformation system in rice by co-cultivating rice calli with Agrobacterium on three filter papers moistened with enriched N6 or DKN media instead of using solid media. Rice calli immersed in Agrobacterium suspension (EHA101, Agrobacterium concentration of OD600=0.04) were co-cultured on three pieces of filter paper (9cm in diameter) moistened with 5.5mL of N6 or DKN liquid co-cultivation medium supplemented with 2,4-d (2mg/L), proline (10mM), casein hydrolysate (300mg/L), sucrose (30g/L), glucose (5g/L), l-cysteine (100mg/L) and acetosyringone (15mg/L) at 25°C for 3 days in the dark. Compared with the transformation efficiency of calli co-cultivated on solid media, transformation efficiency was increased by about fivefold by using the filter paper method for many varieties of rice, including those that previously yielded much poor transformation rates.

  17. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

    PubMed

    Michielse, C B; Arentshorst, M; Ram, A F J; van den Hondel, C A M J J

    2005-01-01

    In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.

  18. Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production.

    PubMed

    Jones, Richard W

    2016-01-01

    When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced.

  19. Plant cell transformation with Agrobacterium tumefaciens under simulated microgravity

    NASA Astrophysics Data System (ADS)

    Sarnatska, Veresa; Gladun, Hanna; Padalko, Svetlana

    To investigate simulated microgravity (clinorotation) effect on plant cell transformation with Agrobacterium tumefaciens and crown gall formation, the culture of primary explants of potato and Jerusalem artichoke tubers was used. It is found that the efficiency of tumor formation and development in clinorotated explants are considerably reduced. When using the explants isolated from potato tubers clinorotated for 3, 5 and 19 days, drastic reduction of formation and development of crown gall tumors was observed. Conversely, the tumor number and their development increased when potato tubers were clinorotated for one day. As was estimated by us previously, cells of Jerusalem artichoke explants are the most sensitive to agrobacteria on 4-5 h of in vitro culturing and this time corresponds to the certain period of G1-stage of the cell cycle. We have also estimated that this period is characterized by the increase of binding of acridine orange by nuclear chromatin and increase in activity of RNA-polymerase I and II. Inoculation of explants with agrobacteria in this period was the most optimal for transformation and crown gall induction. We estimated that at four - hour clinorotation of explants the intensity of acridine orange binding to nuclei was considerably lower than on 4h in the control. At one-day clinorotation of potato tubers, a considerable increase in template accessibility of chromatin and in activity of RNA-polymerase I and II occurred. These results may serve as an evidence for the ability of plant dormant tissues to respond to microgravity. Another demonstration of dormant tissue response to changed gravity we obtained when investigating pathogenesis-related proteins (PR-proteins). PR-proteins were subjected to nondenaturing PAGE.and we have not found any effect of microgravity on PR-proteins of potato explants with normal or tumorous growth. We may suggest that such response derives from the common effects of two stress factors - wounding and changed

  20. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2011-01-01

    VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell. PMID:22028781

  1. Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants.

    PubMed

    Ceasar, S Antony; Ignacimuthu, S

    2011-09-01

    A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM L: -cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R(1) progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R(1) progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.

  2. Characterization of the binding of Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) to glycosphingolipids, using a solid-phase overlay approach

    SciTech Connect

    Stroemberg, N.K.; Karlsson, K.A. )

    1990-07-05

    Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) were radiolabeled externally (125I) or metabolically (35S) and analyzed for their ability to bind glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells. Two binding properties were found and characterized in detail. (i) Both bacteria showed binding to lactosylceramide (LacCer) in a fashion similar to bacteria characterized earlier. The activity of free LacCer was dependent on the ceramide structure; species with 2-hydroxy fatty acid and/or a trihydroxy base were positive, while species with nonhydroxy fatty acid and a dihydroxy base were negative binders. Several glycolipids with internal lactose were active but only gangliotriaosylceramide and gangliotetraosylceramide were as active as free LacCer. The binding to these three species was half-maximal at about 200 ng of glycolipid and was not blocked by preincubation of bacteria with free lactose or lactose-bovine serum albumin. (ii) A. naeslundii, unlike A. viscosus, showed a superimposed binding concluded to be to terminal or internal GalNAc beta and equivalent to a lactose-inhibitable specificity previously analyzed by other workers. Terminal Gal beta was not recognized in several glycolipids, although free Gal and lactose were active as soluble inhibitors. The binding was half-maximal at about 10 ng of glycolipid. A glycolipid mixture prepared from a scraping of human buccal epithelium contained an active glycolipid with sites for both binding specificities.

  3. Efficacy of oral Bifidobacterium bifidum ATCC 29521 on microflora and antioxidant in mice.

    PubMed

    Wang, Bao-gui; Xu, Hai-bo; Xu, Feng; Zeng, Zhe-ling; Wei, Hua

    2016-03-01

    This study aimed to examine whether Bifidobacterium bifidum ATCC 29521, a species of colonic microflora in humans, is involved in the intestinal tract of mice. This study was also conducted to determine the antioxidant activity of this species by evaluating different microbial populations and reactive oxygen species isolated from feces and intestinal contents for 28 days of oral administration. Microbial diversities were assessed through bacterial culture techniques, PCR-DGGE, and real-time PCR. This study showed that the intake of B. bifidum ATCC 29521 significantly (p < 0.05) improved the ecosystem of the intestinal tract of BALB/c mice by increasing the amount of probiotics (Lactobacillus intestinalis and Lactobacillus crispatus) and by reducing unwanted bacterial populations (Enterobacter, Escherichia coli). Antioxidative activities of incubated cell-free extracts were evaluated through various assays, including the scavenging ability of DPPH radical (64.5% and 67.54% (p < 0.05), respectively, at 21 days in nutrients and 28 days in MRS broth), superoxide anion, and hydroxyl radical (85% and 61.5% (p < 0.05), respectively, at intestinal contents in nutrients and 21 days in MRS broth). Total reducing power (231.5 μmol/L (p < 0.05), 14 days in MRS broth) and mRNA level of genes related to oxidative stress were also determined. Results indicated that B. bifidum ATCC 29521 elicits a beneficial effect on murine gut microbiota and antioxidant activities compared with the control samples. This species can be considered as a potential bioresource antioxidant to promote health. Bifidobacterium bifidum ATCC 29521 may also be used as a promising material in microbiological and food applications. PMID:26863255

  4. Complete Genome Sequence of Pseudomonas syringae pv. lapsa Strain ATCC 10859, Isolated from Infected Wheat

    PubMed Central

    Kong, Jun; Jiang, Hongshan; Li, Baiyun; Zhao, Wenjun

    2016-01-01

    Pseudomonas syringae pv. lapsa is a pathovar of Pseudomonas syringae that can infect wheat. The complete genome of P. syringae pv. lapsa strain ATCC 10859 contains a 5,918,899-bp circular chromosome with 4,973 coding sequences, 16 rRNAs, 69 tRNAs, and an average GC content of 59.13%. The analysis of this genome revealed several gene clusters that are related to pathogenesis and virulence. PMID:26941133

  5. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    PubMed

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. PMID:26168906

  6. Complete genome sequence of a malodorant-producing acetogen, Clostridium scatologenes ATCC 25775(T).

    PubMed

    Zhu, Zhengang; Guo, Ting; Zheng, Huajun; Song, Tianshun; Ouyang, Pingkai; Xie, Jingjing

    2015-10-20

    Clostridium scatologenes ATCC 25775(T) is an acetogenic anaerobic bacteria known to be capable of synthesizing volatile fatty acids and solvents from CO2 or CO on its autotrophic mode and producing 3-methylindole and 4-methylphenol on its heterotrophic mode. Here, we report the complete genome sequence of this strain, which might provide a lot of valuable information for developing metabolic engineering strategies to produce biofuels or chemicals from greenhouse gases. PMID:26210291

  7. Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032.

    PubMed

    Wada, Masaru; Sawada, Kazunori; Ogura, Kotaro; Shimono, Yuta; Hagiwara, Takuya; Sugimoto, Masakazu; Onuki, Akiko; Yokota, Atsushi

    2016-02-01

    Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum.

  8. Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

    SciTech Connect

    Aryal, Uma K.; Stockel, Jana; Krovvidi, Ravi K.; Gritsenko, Marina A.; Monroe, Matthew E.; Moore, Ronald J.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2011-12-01

    Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2)-fixation in the dark and photosynthesis in the light. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis reveals fundamental insights into the control and regulation of these functions. To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Analysis of protein functions revealed that the expression of nitrogenase in the dark is mediated by higher respiration and glycogen metabolism. We have also shown that Cyanothece ATCC51142 utilizes alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. In conclusion, this study provides a deeper insight into how Cyanothece ATCC51142 modulates cellular functions to accommodate photosynthesis and N2-fixation within the single cell.

  9. Dynamic proteome analysis of Cyanothece sp. ATCC 51142 under constant light

    SciTech Connect

    Aryal, Uma K.; Stockel, Jana; Welsh, Eric A.; Gritsenko, Marina A.; Nicora, Carrie D.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2012-02-03

    Understanding the dynamic nature of protein abundances provides insights into protein turnover not readily apparent from conventional, static mass spectrometry measurements. This level of data is particularly informative when surveying protein abundances in biological systems subjected to large perturbations or alterations in environment such as cyanobacteria. Our current analysis expands upon conventional proteomic approaches in cyanobacteria by measuring dynamic changes of the proteome using a 13C15N-L-leucine metabolic labeling in Cyanothece ATCC51142. Metabolically labeled Cyanothece ATCC51142 cells grown under nitrogen sufficient conditions in continuous light were monitored longitudinally for isotope incorporation over a 48 h period, revealing 422 proteins with dynamic changes in abundances. In particular, proteins involved in carbon fixation, pentose phosphate pathway, cellular protection, redox regulation, protein folding, assembly and degradation showed higher levels of isotope incorporation suggesting that these biochemical pathways are important for growth under non-diazotrophic conditions. Calculation of relative isotope abundances (RIA) values allowed to measure actual active protein synthesis over time for different biochemical pathways under non-diazotrophic conditions. Overall results demonstrated the utility of 'non-steady state' pulsed metabolic labeling for systems-wide dynamic quantification of the proteome in Cyanothece ATCC51142 that can also be applied to other cyanobacteria.

  10. Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579

    PubMed Central

    Abfalter, Carmen M.; Schönauer, Esther; Ponnuraj, Karthe; Huemer, Markus; Gadermaier, Gabriele; Regl, Christof; Briza, Peter; Ferreira, Fatima; Huber, Christian G.; Brandstetter, Hans; Posselt, Gernot; Wessler, Silja

    2016-01-01

    Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications. PMID:27588686

  11. Genome-scale reconstruction of metabolic networks of Lactobacillus casei ATCC 334 and 12A.

    PubMed

    Vinay-Lara, Elena; Hamilton, Joshua J; Stahl, Buffy; Broadbent, Jeff R; Reed, Jennifer L; Steele, James L

    2014-01-01

    Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications.

  12. Genome-scale reconstruction of metabolic networks of Lactobacillus casei ATCC 334 and 12A.

    PubMed

    Vinay-Lara, Elena; Hamilton, Joshua J; Stahl, Buffy; Broadbent, Jeff R; Reed, Jennifer L; Steele, James L

    2014-01-01

    Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications. PMID:25365062

  13. Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

    PubMed Central

    Gaballa, A; Abeysinghe, P D; Urich, G; Matthijs, S; De Greve, H; Cornelis, P; Koedam, N

    1997-01-01

    Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism. PMID:9361421

  14. Degradation of nitrocellulose-based paint by Desulfovibrio desulfuricans ATCC 13541.

    PubMed

    Giacomucci, L; Toja, F; Sanmartín, P; Toniolo, L; Prieto, B; Villa, F; Cappitelli, F

    2012-09-01

    Nitrocellulose is one of the most commonly used compounds in ammunition and paint industries and its recalcitrance to degradation has a negative impact on human health and the environment. In this study the capability of Desulfovibrio desulfuricans ATCC 13541 to degrade nitrocellulose as binder in paint was assayed for the first time. Nitrocellulose-based paint degradation was followed by monitoring the variation in nitrate, nitrite and ammonium content in the culture medium using Ultraviolet-Visible spectroscopy. At the same time cell counts and ATP assay were performed to estimate bacterial density and activity in all samples. Infrared spectroscopy and colorimetric measurements of paint samples were performed to assess chemical and colour changes due to the microbial action. Microscope observations of nitrocellulose-based paint samples demonstrated the capability of the bacterium to adhere to the paint surface and change the paint adhesive characteristics. Finally, preliminary studies of nitrocellulose degradation pathway were conducted by assaying nitrate- and nitrite reductases activity in D. desulfuricans grown in presence or in absence of paint. We found that D. desulfuricans ATCC 13541 is able to transform nitrocellulose as paint binder and we hypothesised ammonification as degradation pathway. The results suggest that D. desulfuricans ATCC 13541 is a good candidate as a nitrocellulose-degrading bacterium.

  15. Genome –Scale Reconstruction of Metabolic Networks of Lactobacillus casei ATCC 334 and 12A

    PubMed Central

    Vinay-Lara, Elena; Hamilton, Joshua J.; Stahl, Buffy; Broadbent, Jeff R.; Reed, Jennifer L.; Steele, James L.

    2014-01-01

    Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications. PMID:25365062

  16. Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T

    PubMed Central

    Iiyama, Kazuhiro; Mon, Hiroaki; Mori, Kazuki; Mitsudome, Takumi; Lee, Jae Man; Kusakabe, Takahiro; Tashiro, Kousuke; Asano, Shin-ichiro; Yasunaga-Aoki, Chisa

    2015-01-01

    A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706T shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706T were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706T and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage. PMID:25853059

  17. Characterization of enzymatic reduction of hexavalent chromium by Escherichia coli ATCC 33456.

    PubMed Central

    Shen, H; Wang, Y T

    1993-01-01

    Chromium reduction by Escherichia coli ATCC 33456 quantitatively transferred hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III). The reduced chromium was predominantly present in the external medium. Supernatant fluids of cell extract, obtained by centrifugation at 12,000 and 150,000 x g, showed almost the same Cr(VI) reduction activity, indicating that Cr(VI) reduction by E. coli ATCC 33456 was a largely soluble reductase activity. In studies with respiratory inhibitors, no inhibitory effects on aerobic and anaerobic Cr(VI) reduction were demonstrated by addition of cyanide, azide, and rotenone into both intact cell cultures and supernatant fluids of E. coli ATCC 33456. Although cytochromes b and d were identified in the membrane fraction of cell extracts, Cr(VI) was not reduced by the membrane fraction alone. The cytochrome difference spectra analysis also indicated that these cytochromes of the respiratory chain require the presence of the soluble Cr(VI) reductase to mediate electron transport to Cr(VI). Stimulation of Cr(VI) reduction by an uncoupler, 2,4-dinitrophenol, indicated that the respiratory-chain-linked electron transport to Cr(VI) was limited by the rate of dissipation of the proton motive force. PMID:8285683

  18. Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579.

    PubMed

    Abfalter, Carmen M; Schönauer, Esther; Ponnuraj, Karthe; Huemer, Markus; Gadermaier, Gabriele; Regl, Christof; Briza, Peter; Ferreira, Fatima; Huber, Christian G; Brandstetter, Hans; Posselt, Gernot; Wessler, Silja

    2016-01-01

    Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications. PMID:27588686

  19. Inter-organ defense networking: Leaf whitefly sucking elicits plant immunity to crown gall disease caused by Agrobacterium tumefaciens.

    PubMed

    Park, Yong-Soon; Ryu, Choong-Min

    2015-01-01

    Plants have elaborate defensive machinery to protect against numerous pathogens and insects. Plant hormones function as modulators of defensive mechanisms to maintain plant resistance to natural enemies. Our recent study suggests that salicylic acid (SA) is the primary phytohormone regulating plant responses to Agrobacterium tumefaciens infection. Tobacco (Nicotiana benthamiana Domin.) immune responses against Agrobacterium-mediated crown gall disease were activated by exposure to the sucking insect whitefly, which stimulated SA biosynthesis in aerial tissues; in turn, SA synthesized in aboveground tissues systemically modulated SA secretion in root tissues. Further investigation revealed that endogenous SA biosynthesis negatively modulated Agrobacterium-mediated plant genetic transformation. Our study provides novel evidence that activation of the SA-signaling pathway mediated by a sucking insect infestation has a pivotal role in subsequently attenuating Agrobacterium infection. These results demonstrate new insights into interspecies cross-talking among insects, plants, and soil bacteria. PMID:26357873

  20. Inter-organ defense networking: Leaf whitefly sucking elicits plant immunity to crown gall disease caused by Agrobacterium tumefaciens

    PubMed Central

    Park, Yong-Soon; Ryu, Choong-Min

    2015-01-01

    Plants have elaborate defensive machinery to protect against numerous pathogens and insects. Plant hormones function as modulators of defensive mechanisms to maintain plant resistance to natural enemies. Our recent study suggests that salicylic acid (SA) is the primary phytohormone regulating plant responses to Agrobacterium tumefaciens infection. Tobacco (Nicotiana benthamiana Domin.) immune responses against Agrobacterium-mediated crown gall disease were activated by exposure to the sucking insect whitefly, which stimulated SA biosynthesis in aerial tissues; in turn, SA synthesized in aboveground tissues systemically modulated SA secretion in root tissues. Further investigation revealed that endogenous SA biosynthesis negatively modulated Agrobacterium-mediated plant genetic transformation. Our study provides novel evidence that activation of the SA-signaling pathway mediated by a sucking insect infestation has a pivotal role in subsequently attenuating Agrobacterium infection. These results demonstrate new insights into interspecies cross-talking among insects, plants, and soil bacteria. PMID:26357873

  1. Comparative analysis of transgenic tall fescue (Festuca arundinacea Schreb.) plants obtained by Agrobacterium-mediated transformation and particle bombardment.

    PubMed

    Gao, Caixia; Long, Danfeng; Lenk, Ingo; Nielsen, Klaus Kristian

    2008-10-01

    Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants.

  2. Bacteremia due to Agrobacterium tumefaciens (radiobacter). Report of infection in a pregnant women and her stillborn fetus.

    PubMed

    Southern, P M

    1996-01-01

    Agrobacterium tumefaciens (radiobacter) is usually a plant pathogen, but is isolated occasionally from human clinical specimens, frequently along with other bacteria. Agrobacterium tumefaciens (radiobacter) has been isolated from blood, central intravenous catheters, peritoneal fluid, urine, and cellulitis aspirates, often in immunocompromised individuals. This report details the isolation of A. tumefaciens (radiobacter) from the blood of a pregnant woman, as well as from the blood of her stillborn, premature fetus. It is, to our knowledge, the first report of such an occurrence.

  3. Initial reaction(s) in biotransformation of CL-20 is catalyzed by salicylate 1-monooxygenase from Pseudomonas sp. strain ATCC 29352.

    PubMed

    Bhushan, Bharat; Halasz, Annamaria; Spain, Jim C; Hawari, Jalal

    2004-07-01

    CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C(6)H(6)N(12)O(12)), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 +/- 0.011 and 0.043 +/- 0.003 nmol min(-1) mg of protein(-1) under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M - H](-) at 345 Da, corresponding to an empirical formula of C(6)H(6)N(10)O(8), produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M - H](-) at 381 Da corresponding to an empirical formula of C(6)H(10)N(10)O(10). The latter was a hydrated product of the metabolite C(6)H(6)N(10)O(8) with addition of two H(2)O molecules, as confirmed by tests using (18)O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical. PMID:15240281

  4. Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from Pseudomonas sp. Strain ATCC 29352

    PubMed Central

    Bhushan, Bharat; Halasz, Annamaria; Spain, Jim C.; Hawari, Jalal

    2004-01-01

    CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C6H6N12O12), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 ± 0.011 and 0.043 ± 0.003 nmol min−1 mg of protein−1 under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M − H]− at 345 Da, corresponding to an empirical formula of C6H6N10O8, produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M − H]− at 381 Da corresponding to an empirical formula of C6H10N10O10. The latter was a hydrated product of the metabolite C6H6N10O8 with addition of two H2O molecules, as confirmed by tests using 18O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical. PMID:15240281

  5. Arabidopsis VIRE2 INTERACTING PROTEIN2 Is Required for Agrobacterium T-DNA Integration in Plants[W

    PubMed Central

    Anand, Ajith; Krichevsky, Alexander; Schornack, Sebastian; Lahaye, Thomas; Tzfira, Tzvi; Tang, Yuhong; Citovsky, Vitaly; Mysore, Kirankumar S.

    2007-01-01

    Agrobacterium tumefaciens–mediated genetic transformation is an efficient tool for genetic engineering of plants. VirE2 is a single-stranded DNA binding Agrobacterium protein that is transported into the plant cell and presumably protects the T-DNA from degradation. Using a yeast two-hybrid system, we identified Arabidopsis thaliana VIRE2-INTERACTING PROTEIN2 (VIP2) with a NOT domain that is conserved in both plants and animals. Furthermore, we provide evidence supporting VIP2 interaction with VIP1, a basic domain/leucine zipper motif–containing protein required for nuclear import and integration of T-DNA. Virus-induced gene silencing of VIP2 in Nicotiana benthamiana and characterization of the Arabidopsis vip2 mutant (At vip2) demonstrate that VIP2 is required for Agrobacterium-mediated stable transformation but not for transient transformation. Assays based upon a promoter-trap vector and quantification of T-DNA integration further confirmed VIP2 involvement in T-DNA integration. Interestingly, VIP2 transcripts were induced to a greater extent over prolonged periods after infection with a T-DNA transfer-competent Agrobacterium strain compared with the transfer-deficient Agrobacterium strain. Transcriptome analyses of At vip2 suggest that VIP2 is likely a transcriptional regulator, and the recalcitrancy to transformation in At vip2 is probably due to the combination of muted gene expression response upon Agrobacterium infection and repression of histone genes resulting in decreased T-DNA integration events. PMID:17496122

  6. Colonization of arbuscular-mycorrhizal fungi on Ri T-DNA transformed roots in synthetic medium.

    PubMed

    Abdul-Khaliq; Bagyaraj, D J

    2000-11-01

    Hairy root culture of tomato (Lycopersicon esculantum L.) was induced with three strains of Agrobacterium rhizogenes namely A4, ATCC 15834 and LBA 9402. The best response in terms of growth of hairy root was observed with A. rhizogenes strain A4 and LBA 9402 followed by ATCC 15834. Hairy roots were maintained on Murashige and Skoog (MS) medium but it could also grow on minimal (M) medium. Spores of Gigaspora margarita were isolated by wet sieving and decanting method and further recovered by sucrose density gradient method. A new method for surface sterilization of spores has been described which is simpler than the methods described earlier. Surface sterilized spores of G. margarita were used for inoculation of transformed roots grown on M medium as it was found more favourable for germination and growth of spores. During co-cultivation, mycorrhizal spore germination and its penetration into root cortex were observed. Inter and intracellular mycelial spread and formation of arbuscules were also observed in the cortical region of transformed roots of this plant.

  7. A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma.

    PubMed

    Valimehr, Sepideh; Sanjarian, Forough; Sohi, Haleh Hashemi; Sharafi, Ali; Sabouni, Farzaneh

    2014-07-01

    Nepeta pogonosperma is an important medicinal plant with anti-inflammatory effects. An efficient and reliable transformation system for this plant was developed through optimization of several factors which affected the rate of Agrobacterium rhizogenes mediated transformation. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, two explant types, leaves and stems, and several co-cultivation media were examined. The maximum rate of hairy root induction was obtained from stem explants using MSU440 and ATCC15834 bacterial strains. A drastic increase in the frequency of transformation (91 %) was observed when MS medium lacking NH4NO3, KH2PO4, KNO3 and CaCl2. Hairy root lines were confirmed by polymerase chain reaction (PCR) using primers of the rolB gene. According to Southern blot analysis, one T-DNA copy was inserted into each of the hairy root lines. In the present study, transgenic hairy roots have been obtained trough genetic transformation by A. rhizogenes harbouring two plasmids, the Ri plasmid and pBI121 binary vector harbouring gus reporter gene. Expression of the gus gene in transgenic hairy root was confirmed by histochemical GUS assay.

  8. A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma.

    PubMed

    Valimehr, Sepideh; Sanjarian, Forough; Sohi, Haleh Hashemi; Sharafi, Ali; Sabouni, Farzaneh

    2014-07-01

    Nepeta pogonosperma is an important medicinal plant with anti-inflammatory effects. An efficient and reliable transformation system for this plant was developed through optimization of several factors which affected the rate of Agrobacterium rhizogenes mediated transformation. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, two explant types, leaves and stems, and several co-cultivation media were examined. The maximum rate of hairy root induction was obtained from stem explants using MSU440 and ATCC15834 bacterial strains. A drastic increase in the frequency of transformation (91 %) was observed when MS medium lacking NH4NO3, KH2PO4, KNO3 and CaCl2. Hairy root lines were confirmed by polymerase chain reaction (PCR) using primers of the rolB gene. According to Southern blot analysis, one T-DNA copy was inserted into each of the hairy root lines. In the present study, transgenic hairy roots have been obtained trough genetic transformation by A. rhizogenes harbouring two plasmids, the Ri plasmid and pBI121 binary vector harbouring gus reporter gene. Expression of the gus gene in transgenic hairy root was confirmed by histochemical GUS assay. PMID:25049462

  9. Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp.

    PubMed

    Cha, Thye-San; Chen, Chin-Fong; Yee, Willy; Aziz, Ahmad; Loh, Saw-Hong

    2011-03-01

    The use of acetosyringone in Agrobacterium-mediated gene transfer into plant hosts has been favored for the past few decades. The influence of other phenolic compounds and their effectiveness in Agrobacterium-mediated plant transformation systems has been neglected. In this study, the efficacy of four phenolic compounds on Agrobacterium-mediated transformation of the unicellular green alga Nannochloropsis sp. (Strain UMT-M3) was assessed by using β-glucuronidase (GUS) assay. We found that cinnamic acid, vanillin and coumarin produced higher percentages of GUS positive cells as compared to acetosyringone. These results also show that the presence of methoxy group in the phenolic compounds may not be necessary for Agrobacterium vir gene induction and receptor binding as suggested by previous studies. These findings provide possible alternative Agrobacterium vir gene inducers that are more potent as compared to the commonly used acetosyringone in achieving high efficiency of Agrobacterium-mediated transformation in microalgae and possibly for other plants.

  10. Characterization of an Unusual New Agrobacterium tumefaciens Strain from Chrysanthemum morifolium Ram †

    PubMed Central

    Bush, Arla L.; Pueppke, Steven G.

    1991-01-01

    We characterized five isolates of Agrobacterium tumefaciens from naturally occurring galls on Chrysanthemum morifolium. The isolates are similar, possibly identical, members of a single strain of A. tumefaciens that we designate Chry5. The strain is a biotype I, as indicated by its response to both newly described and traditional biotype tests. Chry5 produces tumors on at least 10 plant species. It is unusual in its ability to form efficiently large tumors on soybean (Glycine max), a species normally refractory to transformation. Chry5 is unable to utilize octopine or mannopine as a carbon source. Although Chry5 can catabolize a single isomer each of nopaline and succinamopine, it differs from other known nopaline and succinamopine strains in its insensitivity to agrocin 84. This pattern of opine catabolism is unique among Agrobacterium strains examined to date. All five isolates of Chry5 contain at least two plasmids, one of which shares homology with pTiB6. Images PMID:16348549

  11. Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Xue, Chunsheng; Wu, Dongliang; Condon, Bradford J; Bi, Qing; Wang, Weiwei; Turgeon, B Gillian

    2013-06-01

    Setosphaeria turcica, a hemibiotrophic pathogenic dothideomycete, is the causal agent of Northern Leaf Blight of maize, which periodically causes significant yield losses worldwide. To explore molecular mechanisms of fungal pathogenicity and virulence to the host, an efficient targeted gene knockout transformation system using Agrobacterium tumefaciens was established with field collected strains. The starting materials, incubation time, induction medium type, Agrobacterium cell density, and method of co-incubation were optimized for deletion of 1,3,8-trihydroxynaphthalene reductase, a gene in the melanin biosynthesis pathway, as a test case. Four additional genes were deleted in two different S. turcica field isolates to confirm robustness of the method. One of these mutant strains was reduced in virulence compared with the wild-type strain when inoculated on susceptible maize. Transformation efficiency was ≈20 ± 3 transformants per 1× 10(6) germlings and homologous recombination efficiency was 33.3 to 100%. PMID:23384859

  12. Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake.

    PubMed

    Gupta, V; Lakshmi Sita, G; Shaila, M S; Jagannathan, V

    1993-05-01

    Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes. PMID:24197344

  13. Regeneration and Agrobacterium-mediated transformation of hop (Humulus lupulus L.).

    PubMed

    Horlemann, C; Schwekendiek, A; Höhnle, M; Weber, G

    2003-10-01

    An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (beta-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%. PMID:12898178

  14. Multiple host-cell recombination pathways act in Agrobacterium-mediated transformation of plant cells.

    PubMed

    Mestiri, Imen; Norre, Frédéric; Gallego, Maria E; White, Charles I

    2014-02-01

    Using floral-dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non-homologous and homologous recombination pathways in Agrobacterium-mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium-mediated transformation. Although integration of T-DNA into the plant genome is severely compromised in the absence of known DNA double-strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T-DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation.

  15. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    NASA Technical Reports Server (NTRS)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

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

  16. Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda.

    PubMed

    Stevens, Micah E; Pijut, Paula M

    2014-06-01

    This transformation and regeneration protocol provides an integral framework for the genetic improvement of Fraxinus profunda (pumpkin ash) for future development of plants resistant to the emerald ash borer. Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to eliminate native Fraxinus spp. from the natural landscape. Hypocotyls were successfully transformed with Agrobacterium strain EHA105 harboring the pq35GR vector, containing an enhanced green fluorescent protein (EGFP) as well as a fusion gene between neomycin phosphotransferase (nptII) and gusA. Hypocotyls were cultured for 7 days on Murashige and Skoog (MS) medium with 22.2 μM 6-benzyladenine (BA), 4.5 μM thidiazuron (TDZ), 50 mg L(-1) adenine hemisulfate (AS), and 10 % coconut water (CW) prior to transformation. Hypocotyls were transformed using 90 s sonication plus 10 min vacuum infiltration after Agrobacterium was exposed to 100 μM acetosyringone for 1 h. Adventitious shoots were regenerated on MS medium with 22.2 μM BA, 4.5 μM TDZ, 50 mg L(-1) AS, 10 % CW, 400 mg L(-1) timentin, and 20 mg L(-1) kanamycin. Timentin at 400 and 20 mg L(-1) kanamycin were most effective at controlling Agrobacterium growth and selecting for transformed cells, respectively. The presence of nptII, GUS (β-glucuronidase), and EGFP in transformed plants was confirmed using polymerase chain reaction (PCR), while the expression of EGFP was also confirmed through fluorescent microscopy and reverse transcription-PCR. This transformation protocol provides an integral foundation for future genetic modifications of F. profunda to provide resistance to EAB. PMID:24493252

  17. Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains.

    PubMed Central

    Steck, T R; Kado, C I

    1990-01-01

    Certain virulence region operons of the Agrobacterium tumefaciens Ti plasmid promoted conjugative Ti plasmid transfer. Mutations in the vir region of pTiC58 inhibited conjugative plasmid transfer between A. tumefaciens strains. Mutations in virA, virG, 5' virB, and virE had the greatest effect on plasmid transfer, and mutations in virC had no effect. Transfer inhibition in vir mutants occurred in the presence or absence of acetosyringone. PMID:2318813

  18. Role of Agrobacterium virB genes in transfer of T complexes and RSF1010.

    PubMed

    Fullner, K J

    1998-01-01

    Nonpolar virB mutants of Agrobacterium tumefaciens were tested for RSF1010 mobilization and extracellular complementation. virB2 to virB11 were essential for transfer in both assays. virB1 was essential only for high frequency transfer of RSF1010 and VirE2. Coordinated transfer of a preassembled T complex is supported by these data and competition studies. PMID:9440537

  19. Genome sequence of the nicotine-degrading Agrobacterium tumefaciens S33.

    PubMed

    Yu, Wenjun; Li, Huili; Xie, Kebo; Huang, Haiyan; Xie, Huijun; Wang, Shuning

    2016-06-20

    Agrobacterium tumefaciens S33 is capable of growing with nicotine as the sole source of carbon and nitrogen, and has the potential to dispose of tobacco wastes and transform nicotine into functionalized pyridines intermediates, which are important precursors for some valuable drugs and insecticides. Here we report the complete genome sequence of strain S33 and predict the gene cluster involved in nicotine catabolism according to the annotation. PMID:27106695

  20. Detection of Activity Responsible for Induction of the Agrobacterium tumefaciens Virulence Genes in Bacteriological Agar.

    PubMed

    Loubens, I; Chilton, W S; Dion, P

    1997-11-01

    Agrobacterium tumefaciens C58 grown on acidic medium containing glucose and solidified with bacteriological agar expressed a virB::lacZ fusion. No expression of this fusion was observed on a similar medium which was solidified with purified agarose. The fraction from bacteriological agar which was responsible for vir gene induction was extracted with methanol and partially purified by preparative thin-layer chromatography. PMID:16535740

  1. Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

    PubMed Central

    Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

    2014-01-01

    The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

  2. Catheter-related bacteremia caused by Agrobacterium radiobacter in a hemodialysis patient.

    PubMed

    Hanada, Shigeru; Iwamoto, Mari; Kobayashi, Namiko; Ando, Ryoichi; Sasaki, Sei

    2009-01-01

    Agrobacterium radiobacter, a Gram-negative bacillus, is recognized as an emerging opportunistic human pathogen that has a propensity to cause infections in patients with indwelling foreign devices. Here, we describe the first reported case of catheter-related bacteremia caused by A. radiobacter in a hemodialysis patient with a long-term tunneled-cuffed hemodialysis catheter. This case shows that A. radiobacter should be included in the list of pathogens that can cause catheter-related bacteremia in hemodialysis patients.

  3. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    PubMed

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  4. Investigation of the Amycolatopsis sp. strain ATCC 39116 vanillin dehydrogenase and its impact on the biotechnical production of vanillin.

    PubMed

    Fleige, Christian; Hansen, Gunda; Kroll, Jens; Steinbüchel, Alexander

    2013-01-01

    The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDH(ATCC 39116)). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDH(ATCC 39116) was purified to apparent electrophoretic homogeneity and exhibited NAD(+)-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin.

  5. Investigation of the Amycolatopsis sp. strain ATCC 39116 vanillin dehydrogenase and its impact on the biotechnical production of vanillin.

    PubMed

    Fleige, Christian; Hansen, Gunda; Kroll, Jens; Steinbüchel, Alexander

    2013-01-01

    The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDH(ATCC 39116)). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDH(ATCC 39116) was purified to apparent electrophoretic homogeneity and exhibited NAD(+)-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin. PMID:23064333

  6. Use of Ti Plasmid DNA Probes for Determining Tumorigenicity of Agrobacterium Strains

    PubMed Central

    Burr, Thomas J.; Norelli, John L.; Katz, Barbara H.; Bishop, Andrew L.

    1990-01-01

    Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two 32P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 106 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains. Images PMID:16348218

  7. Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsis thaliana.

    PubMed

    Lee, Chil-Woo; Efetova, Marina; Engelmann, Julia C; Kramell, Robert; Wasternack, Claus; Ludwig-Müller, Jutta; Hedrich, Rainer; Deeken, Rosalia

    2009-09-01

    Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis-Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria. PMID:19794116

  8. Agrobacterium tumefaciens Gene Transfer: How a Plant Pathogen Hacks the Nuclei of Plant and Nonplant Organisms.

    PubMed

    Bourras, Salim; Rouxel, Thierry; Meyer, Michel

    2015-10-01

    Agrobacterium species are soilborne gram-negative bacteria exhibiting predominantly a saprophytic lifestyle. Only a few of these species are capable of parasitic growth on plants, causing either hairy root or crown gall diseases. The core of the infection strategy of pathogenic Agrobacteria is a genetic transformation of the host cell, via stable integration into the host genome of a DNA fragment called T-DNA. This genetic transformation results in oncogenic reprogramming of the host to the benefit of the pathogen. This unique ability of interkingdom DNA transfer was largely used as a tool for genetic engineering. Thus, the artificial host range of Agrobacterium is continuously expanding and includes plant and nonplant organisms. The increasing availability of genomic tools encouraged genome-wide surveys of T-DNA tagged libraries, and the pattern of T-DNA integration in eukaryotic genomes was studied. Therefore, data have been collected in numerous laboratories to attain a better understanding of T-DNA integration mechanisms and potential biases. This review focuses on the intranuclear mechanisms necessary for proper targeting and stable expression of Agrobacterium oncogenic T-DNA in the host cell. More specifically, the role of genome features and the putative involvement of host's transcriptional machinery in relation to the T-DNA integration and effects on gene expression are discussed. Also, the mechanisms underlying T-DNA integration into specific genome compartments is reviewed, and a theoretical model for T-DNA intranuclear targeting is presented. PMID:26151736

  9. [Methods for the detection of Agrobacterium from plant, soil and water samples].

    PubMed

    Alippi, Adriana M; López, Ana C; Balatti, Pedro A

    2011-01-01

    The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly difficult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identification techniques by using a set of semi-selective and differential culture media in combination with a specific PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specific PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes. PMID:22274826

  10. Agrobacterium induces expression of a host F-box protein required for tumorigenicity

    PubMed Central

    Zaltsman, Adi; Krichevsky, Alexander; Loyter, Abraham; Citovsky, Vitaly

    2010-01-01

    In plant-pathogen interactions, the host defends against the invading pathogen and the pathogen aims to suppress or subvert this defense. Whereas the defense suppression strategy is relatively well understood for many pathogens, the mechanisms by which pathogens can actively utilize the defense machinery of the host remain obscure. We report that Agrobacterium, a microorganism that elicits neoplastic growths on many plant species, induces expression of a plant defense-related F-box protein, VBF, which it incorporates into its own pathway for genetic transformation. Our data suggest that VBF may function to uncoat the bacterial transferred DNA from its associated virulence VirE2 and host VIP1 proteins via the SCFVBF pathway. Suppression of VBF elevates the intracellular content of VIP1, but renders the plant largely resistant to Agrobacterium, indicating that, in the infection pathway, VBF is functionally epistatic to VIP1. When expressed in Agrobacterium and exported into the plant cell, VBF facilitates tumor formation. PMID:20227663

  11. Transfer of T-DNA from Agrobacterium to the plant cell.

    PubMed

    Zupan, J R; Zambryski, P

    1995-04-01

    Agrobacterium tumefaciens is the causative agent of crown gall, a disease of dicotyledonous plants characterized by a tumorous phenotype. Earlier in this century, scientific interest in A. tumefaciens was based on the possibility that the study of plant tumors might reveal mechanisms that were also operating in animal neoplasia. In the recent past, the tumorous growth was shown to result from the expression of genes coded for by a DNA segment of bacterial origin that was transferred and became stably integrated into the plant genome. This initial molecular characterization of the infection process suggested that Agrobacterium might be used to deliver genetic material into plants. The potential to genetically engineer plants generated renewed interest in the study of A. tumefaciens. In this review, we concentrate on the most recent advances in the study of Agrobacterium-mediated gene transfer, its relationship to conjugation, DNA processing and transport, and nuclear targeting. In the following discussion, references for earlier work can be found in more comprehensive reviews (Hooykaas and Schilperoort, 1992; Zambryski, 1992; Hooykaas and Beijersbergen, 1994). PMID:7770515

  12. Use of Ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains

    SciTech Connect

    Burr, T.J.; Norelli, J.L.; Katz, B.H.; Bishop, A.L. )

    1990-06-01

    Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two {sup 32}P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10{sup 6} CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains.

  13. [Meristematic characteristics of tumors initiated by Agrobacterium tumefaciens in pea plants].

    PubMed

    Vinogradova, A P; Lebedeva, M A; Lutova, L A

    2015-01-01

    It is known that two key groups of plant hormones--auxins and cytokinins--play an important role in plant tumor development. The formation of Agrobacterium-induced tumors results from the horizontal transfer of bacterial oncogenes involved in the biosynthesis of these hormones in the plant genome. The role of transcriptional factors in plant tumor development is poorly investigated. It can be assumed that tumor development associated with abnormal cell proliferation can be controlled by the same set of transcription factors that control normal cell proliferation and, in particular, transcription factors that regulate meristem activity. In the present study, we analyzed the histological organization and distribution of proliferating cells in tumors induced by Agrobacterium tumefaciens on pea hypocotyls. In addition, the expression of a set of meristem-specific genes with Agrobacterium tumefaciens-induced tumor development was analyzed. In general, our results indicate that meristematic structures are present in A. tumefaciens-induced tumors and that the development of such tumors is associated with increased expression of a key gene regulating the root apical meristem--the WOX5 gene.

  14. The Arabidopsis Myb transcription factor MTF1 is a unidirectional regulator of susceptibility to Agrobacterium.

    PubMed

    Sardesai, Nagesh; Laluk, Kristin; Mengiste, Tesfaye; Gelvin, Stanton

    2014-04-30

    We recently described the Arabidopsis Myb transcription factor MTF1 that negatively regulates plant susceptibility to Agrobacterium-mediated transformation. Roots of mtf1 mutant plants show increased susceptibility to several Agrobacterium strains, and complementing the mutants with a MTF1 cDNA decreases transformation susceptibility to wild-type levels. Here, we show that overexpression of MTF1 in a wild-type Arabidopsis background does not result in altered transformation susceptibility. However, MTF1 overexpressing plants show increased root length and larger and darker leaves, indicating that MTF1 plays a role in plant growth and development. MTF1 decreases Arabidopsis root susceptibility specifically to Agrobacterium but plant responses to the pathogens Alternaria brassicicola or Pseudomonas syringae pv Tomato were not altered. However, the homozygous MTF1 mutant mtf1-4 is resistant to Botrytis cinerea strain BO5-10 and is regulated through the ethylene signaling pathway mediated by upregulation of the AP2/ERF transcription factor ORA59. PMID:24785741

  15. [Meristematic characteristics of tumors initiated by Agrobacterium tumefaciens in pea plants].

    PubMed

    Vinogradova, A P; Lebedeva, M A; Lutova, L A

    2015-01-01

    It is known that two key groups of plant hormones--auxins and cytokinins--play an important role in plant tumor development. The formation of Agrobacterium-induced tumors results from the horizontal transfer of bacterial oncogenes involved in the biosynthesis of these hormones in the plant genome. The role of transcriptional factors in plant tumor development is poorly investigated. It can be assumed that tumor development associated with abnormal cell proliferation can be controlled by the same set of transcription factors that control normal cell proliferation and, in particular, transcription factors that regulate meristem activity. In the present study, we analyzed the histological organization and distribution of proliferating cells in tumors induced by Agrobacterium tumefaciens on pea hypocotyls. In addition, the expression of a set of meristem-specific genes with Agrobacterium tumefaciens-induced tumor development was analyzed. In general, our results indicate that meristematic structures are present in A. tumefaciens-induced tumors and that the development of such tumors is associated with increased expression of a key gene regulating the root apical meristem--the WOX5 gene. PMID:25857193

  16. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. PMID:23821951

  17. Mapping of the Interaction Between Agrobacterium tumefaciens and Vanda Kasem's Delight Orchid Protocorm-Like Bodies.

    PubMed

    Gnasekaran, Pavallekoodi; Subramaniam, Sreeramanan

    2015-09-01

    Physical contact between A. tumefaciens and the target plant cell walls is essential to transfer and integrate the transgene to introduce a novel trait. Chemotaxis response and attachment of Agrobacterium towards Vanda Kasem's Delight (VKD) protocorm-like bodies (PLBs) were studied to analyse the interaction between Agrobacterium and PLB during the transformation event. The study shows that initially A. tumefaciens reversibly attached to PLB surface via polar and lateral mode of adherence followed by the irreversible attachment which involved the production of cellulosic fibril by A. tumefaciens. Cellulosic fibril allows formation of biofilm at the tip of trichome. Contrarily, attachment mutant Escherichia coli strain DH5α was significantly deficient in the attachment process. Spectrophotometric GUS assay showed the mean value of attachment by A. tumefaciens was 8.72 % compared to the negative control E. coli strain DH5α that produced 0.16 %. A. tumefaciens swarmed with sharper and brighter edge when severe wounding was applied to the PLBs producing the highest swarming ratio of 1.46 demonstrating the positive effect of the plant exudates on bacterial movement. The study shows that VKD's PLBs are the suitable explants for Agrobacterium-mediated transformation since the bacteria expressed higher competency rate.

  18. Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration.

    PubMed

    Subramanyam, Kondeti; Subramanyam, Koona; Sailaja, K V; Srinivasulu, M; Lakshmidevi, K

    2011-03-01

    A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 μM acetosyringone followed by co-cultivation in 50 μM acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana.

  19. Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat.

    PubMed

    Ding, Liping; Li, Shengchun; Gao, Jianming; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

    2009-01-01

    Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase II, (npt II) and beta-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23-25 degrees C for 3 h, and then co-culturing with Agrobacterium under desiccating condition in the dark at 23-24 degrees C for 2-3 days. Complete analysis of transgene insertion demonstrated that the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable.

  20. Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.

    PubMed

    Tang, Wei; Lin, Jinxing; Newton, Ronald J

    2007-05-01

    Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.

  1. An efficient regeneration protocol for Agrobacterium-mediated transformation of melon (Cucumis melo L.).

    PubMed

    Zhang, H J; Gao, P; Wang, X Z; Luan, F S

    2014-01-08

    An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation, using cotyledon node zone-stem connection region of melon, has been developed. The new Agrobacterium-mediated transformation methodology, independent of organ culture, used the entire germinated seed as explants. The transformation system was maximized to maintain the integrity of melon itself, thus avoiding the limitations of traditional tissue culture methods. The transformation was carried out under a non-sterile environment. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed by PCR and Southern blot analyses. The transformation frequency based on the PCR was 13%. Transgenic melon plants were usually detected by PCR in less than 1 month after Agrobacterium inoculation, and seeds could be harvested in 3 months. The growth characteristics and morphology of the transgenic plants were identical to the untransformed wild-type plants. This method would be beneficial for facilitating the characteristics of gene functions and for boosting the manipulation of melon transformation for commercial purposes.

  2. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  3. Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

    PubMed

    Sivamani, Elumalai; Li, Xianggan; Nalapalli, Samson; Barron, Yoshimi; Prairie, Anna; Bradley, David; Doyle, Michele; Que, Qiudeng

    2015-12-01

    Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments.

  4. Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp.

    PubMed Central

    Fuchs, R L; Keister, D L

    1980-01-01

    Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium. GSII activity disappeared within one generation of growth in two of the strains, but the disappearance of GSII activity required two generations in another. Isoactivity points for transferase assay, which were derived from the pH curves of adenylylated GSI and deadenylylated GSI, were approximately pH 7.8 for both R. trifolii and A. tumefaciens. No isoactivity point was found for R. japonicum under the standard assay conditions used. When the feedback inhibitor glycine was used to inhibit differentially the adenylylated GSI and deadenylylated GSI of R. japonicum, an isoactivity point was observed at pH 7.3. Thus, the transferase activity of GSI could be determined independent of the state of adenylation. A survey of 23 strains of bacteria representing 11 genera indicated that only Rhizobium spp. and Agrobacterium spp. contained GSII. Thus, this enzyme appears to be unique for the Rhizobiaceae. PMID:6107288

  5. Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees.

    PubMed

    López, M M; Gorris, M T; Salcedo, C I; Montojo, A M; Miró, M

    1989-03-01

    The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr against the sensitive strain but was similar against the resistant strain.

  6. Improved penicillin amidase production using a genetically engineered mutant of escherichia coli ATCC 11105

    SciTech Connect

    Robas, N.; Zouheiry, H.; Branlant, G.; Branlant, C. )

    1993-01-05

    Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, the authors constructed various recombinant E. coli HB 101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic acid (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the HindIII fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.

  7. AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

    PubMed Central

    2014-01-01

    Background Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous

  8. Female Reproductive Tissues Are the Primary Target of Agrobacterium-Mediated Transformation by the Arabidopsis Floral-Dip Method1

    PubMed Central

    Desfeux, Christine; Clough, Steven J.; Bent, Andrew F.

    2000-01-01

    The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding β-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved. PMID:10889238

  9. Evaluating Chemical Mitigation of Salmonella Typhimurium ATCC 14028 in Animal Feed Ingredients.

    PubMed

    Cochrane, Roger A; Huss, Anne R; Aldrich, Gregory C; Stark, Charles R; Jones, Cassandra K

    2016-04-01

    Salmonella Typhimurium is a potential feed safety hazard in animal feed ingredients. Thermal mitigation of Salmonella spp. during rendering is effective but does not eliminate the potential for cross-contamination. Therefore, the objective of this experiment was to evaluate the effectiveness of chemicals to mitigate postrendering Salmonella Typhimurium ATCC 14028 contamination in rendered proteins over time. Treatments were arranged in a 6 × 4 factorial with six chemical treatments and four rendered protein meals. The chemical treatments included (i) control without chemical treatment, (ii) 0.3% commercial formaldehyde product, (iii) 2% essential oil blend, (iv) 2% medium chain fatty acid blend, (v) 3% organic acid blend, and (vi) 1% sodium bisulfate. The four rendered protein meals included (i) feather meal, (ii) blood meal, (iii) meat and bone meal, and (iv) poultry by-product meal. After matrices were chemically treated, they were inoculated with Salmonella Typhimurium ATCC 14028, stored at room temperature, and enumerated via plate counts on days 0, 1, 3, 7, 14, 21, and 42 postinoculation. The Salmonella concentration in ingredients treated with medium chain fatty acid and commercial formaldehyde were similar to one another (P = 0.23) but were 2 log lower than the control (P < 0.05). Ingredients treated with organic acids and essential oils also had lower Salmonella concentrations than the control (P < 0.05). Time also played a significant role in Salmonella mitigation, because all days except days 14 and 21 (P = 0.92) differed from one another. Rendered protein matrix also affected Salmonella stability, because concentrations in meat and bone meal and blood meal were similar to one another (P = 0.36) but were greater than levels in feather meal and poultry by-product meal (P < 0.05). In summary, chemical treatment and time both mitigated Salmonella Typhimurium ATCC 14028, but their effectiveness was matrix dependent. Time and chemical treatment with medium

  10. Multicenter Investigation of Gepotidacin (GSK2140944) Agar Dilution Quality Control Determinations for Neisseria gonorrhoeae ATCC 49226.

    PubMed

    Jones, Ronald N; Fedler, Kelley A; Scangarella-Oman, Nicole E; Ross, James E; Flamm, Robert K

    2016-07-01

    Gepotidacin, a novel triazaacenaphthylene antibacterial agent, is the first in a new class of type IIA topoisomerase inhibitors with activity against many biothreat and conventional pathogens, including Neisseria gonorrhoeae To assist ongoing clinical studies of gepotidacin to treat gonorrhea, a multilaboratory quality assurance investigation determined the reference organism (N. gonorrhoeae ATCC 49226) quality control MIC range to be 0.25 to 1 μg/ml (88.8% of gepotidacin MIC results at the 0.5 μg/ml mode). PMID:27161642

  11. Complete genome sequence of Vibrio alginolyticus ATCC 33787(T) isolated from seawater with three native megaplasmids.

    PubMed

    Wang, Pengxia; Wen, Zhongling; Li, Baiyuan; Zeng, Zhenshun; Wang, Xiaoxue

    2016-08-01

    Vibrio alginolyticus, an opportunistic pathogen, is commonly associated with vibriosis in fish and shellfish and can also cause superficial and ear infections in humans. V. alginolyticus ATCC 33787(T) was originally isolated from seawater and has been used as one of the type strains for exploring the virulence factors of marine bacteria and for developing vaccine against vibriosis. Here we sequenced and assembled the whole genome of this strain, and identified three megaplasmids and three Type VI secretion systems, thus providing useful information for the study of virulence factors and for the development of vaccine for Vibrio.

  12. Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein

    PubMed Central

    Lacroix, Benoît; Citovsky, Vitaly

    2015-01-01

    During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF. PMID:26586289

  13. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

    NASA Technical Reports Server (NTRS)

    Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

    1999-01-01

    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.

  14. Production of fructosyltransferase by Aureobasidium sp. ATCC 20524 in batch and two-step batch cultures.

    PubMed

    Salinas, Martín A; Perotti, Nora I

    2009-01-01

    A comparison of fructosyltransferase (EC 2.4.1.9) production by Aureobasidium sp. ATCC 20524 in batch and two step batch cultures was investigated in a 1-l stirred tank reactor using a sucrose supply of 200 g/l. Results showed that the innovative cultivation in two step of Aureobasidium sp. produced more fructosyltransferase (FFase) than the single batch culture at the same sucrose concentration with a maximal enzyme production of 523 U/ml, which was 80.5% higher than the one obtained in the batch culture. The production of fructooligosaccharides (FOSs) was also analyzed; their concentration reached a maximum value of 160 g/l the first day in the two-step culture and 127 g/l in the single-batch mode. The use of the two-step batch culture with Aureobasidium sp. ATCC 20524 in allowing the microorganism to grow up prior to the induction of sucrose (second step), proved to be a powerful method for producing fructosyltransferase and FOSs. PMID:18810518

  15. An Evaluation of Kinetic Models in the Biodesulfurization of Synthetic Oil by Rhodococcus erythropolis ATCC 4277.

    PubMed

    Maass, D; Mayer, D A; Moritz, D E; Oliveira, D; de Souza, A A Ulson; Souza, S M A Guelli

    2015-10-01

    Biodesulfurization is an eco-friendly technology applied in the removal of sulfur from fossil fuels. This technology is based on the use of microorganisms as biocatalysts to convert the recalcitrant sulfur compounds into others easily treatable, as sulfides. Despite it has been studied during the last decades, there are some unsolved questions, as per example the kinetic model which appropriately describes the biodesulfurization globally. In this work, different kinetic models were tested to a batch desulfurization process using dibenzothiophene (DBT) as a model compound, n-dodecane as organic solvent, and Rhodococcus erythropolis ATCC 4277 as biocatalyst. The models were solved by ODE45 function in the MATLAB. Monod model was capable to describe the biodesulfurization process predicting all experimental data with a very good fitting. The coefficients of determination achieved to organic phase concentrations of 20, 80, and 100 % (v/v) were 0.988, 0.995, and 0.990, respectively. R. erythropolis ATCC 4277 presented a good affinity with the substrate (DBT) since the coefficients of saturation obtained to reaction medium containing 20, 80, and 100 % (v/v) were 0.034, 0.07, and 0.116, respectively. This kinetic evaluation provides an improvement in the development of biodesulfurization technology because it showed that a simple model is capable to describe the throughout process. PMID:26201481

  16. Cloning, expression, and sequencing of a protease gene from Bacteroides forsythus ATCC 43037 in Escherichia coli.

    PubMed Central

    Saito, T; Ishihara, K; Kato, T; Okuda, K

    1997-01-01

    We have isolated and characterized an N-benzoyl-Val-Gly-Arg-p-nitroanilide-specific protease gene, designated prtH, from Bacteroides forsythus ATCC 43037. Nucleotide sequencing of the DNA insert from the clone (hereafter referred to as clone FST) revealed that the protease activity corresponded to an open reading frame consisting of 1,272 bp coding for a 47.8-kDa protein. When plasmid pFST was used as a probe in Southern hybridization, Sau3AI-digested chromosomal DNA of B. forsythus ATCC 43037 as well as the chromosomal DNAs of the isolated strains Ta4, TR5, and YG2 showed 0.6- and 0.8-kb hybridizing bands. The cell-free extracts of clone FST showed hemolytic activity on human blood cells. The hydrolytic activity of cell extracts of the pFST clone was inhibited by p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride, leupeptin, N-ethylmaleimide, iodoacetic acid, iodoaceteamide, and EDTA. PMID:9353083

  17. Ferulic acid transformation into the main vanilla aroma compounds by Amycolatopsis sp. ATCC 39116.

    PubMed

    Pérez-Rodríguez, Noelia; Oliveira, Ricardo Pinheiro de Souza; Agrasar, Ana María Torrado; Domínguez, José Manuel

    2016-02-01

    The wild strain Amycolatopsis sp. ATCC 39116 was explored in ferulic acid-based media to produce naturally the aroma components of the cured vanilla pod, namely vanillin,vanillic acid, and vanillyl alcohol. Other phenolic compounds(4-vinyl guaiacol, guaiacol, and protocatechuic acid) were also evaluated. The influence of medium composition,fermentation technology (batch or fed-batch), supplementation with vanillic acid, and inoculum concentration on ferulic acid biotransformation were evaluated. The results postulate the initial concentration of cell mass as the variable with the strongest impact on ferulic acid metabolization under the studied conditions. The highest amounts of vanillin and vanillic acid were achieved at intermediate values of cell mass.Vanillyl alcohol and protocatechuic acid were more closely linked to high cell mass concentrations. Conversely, 4-vinyl guaiacol reached its highest amount at the lowest amount of cell mass. Guaiacol was not detected in any case. Therefore,the initial cell concentration must be considered a critical parameter when using Amycolaptosis sp. ATCC 39116 for the production of vanillin and related compounds. PMID:26476645

  18. Genome Sequence of Aeromonas hydrophila ATCC 7966T: Jack of All Trades▿

    PubMed Central

    Seshadri, Rekha; Joseph, Sam W.; Chopra, Ashok K.; Sha, Jian; Shaw, Jonathan; Graf, Joerg; Haft, Daniel; Wu, Martin; Ren, Qinghu; Rosovitz, M. J.; Madupu, Ramana; Tallon, Luke; Kim, Mary; Jin, Shaohua; Vuong, Hue; Stine, O. Colin; Ali, Afsar; Horneman, Amy J.; Heidelberg, John F.

    2006-01-01

    The complete genome of Aeromonas hydrophila ATCC 7966T was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966T. Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments. PMID:16980456

  19. In silico analysis of Clostridium acetobutylicum ATCC 824 metabolic response to an external electron supply.

    PubMed

    Gallardo, Roberto; Acevedo, Alejandro; Quintero, Julián; Paredes, Ivan; Conejeros, Raúl; Aroca, Germán

    2016-02-01

    The biological production of butanol has become an important research field and thanks to genome sequencing and annotation; genome-scale metabolic reconstructions have been developed for several Clostridium species. This work makes use of the iCAC490 model of Clostridium acetobutylicum ATCC 824 to analyze its metabolic capabilities and response to an external electron supply through a constraint-based approach using the Constraint-Based Reconstruction Analysis Toolbox. Several analyses were conducted, which included sensitivity, production envelope, and phenotypic phase planes. The model showed that the use of an external electron supply, which acts as co-reducing agent along with glucose-derived reducing power (electrofermentation), results in an increase in the butanol-specific productivity. However, a proportional increase in the butyrate uptake flux is required. Besides, the uptake of external butyrate leads to the coupling of butanol production and growth, which coincides with results reported in literature. Phenotypic phase planes showed that the reducing capacity becomes more limiting for growth at high butyrate uptake fluxes. An electron uptake flux allows the metabolism to reach the growth optimality line. Although the maximum butanol flux does not coincide with the growth optimality line, a butyrate uptake combined with an electron uptake flux would result in an increased butanol volumetric productivity, being a potential strategy to optimize the production of butanol by C. acetobutylicum ATCC 824.

  20. Ferulic acid transformation into the main vanilla aroma compounds by Amycolatopsis sp. ATCC 39116.

    PubMed

    Pérez-Rodríguez, Noelia; Oliveira, Ricardo Pinheiro de Souza; Agrasar, Ana María Torrado; Domínguez, José Manuel

    2016-02-01

    The wild strain Amycolatopsis sp. ATCC 39116 was explored in ferulic acid-based media to produce naturally the aroma components of the cured vanilla pod, namely vanillin,vanillic acid, and vanillyl alcohol. Other phenolic compounds(4-vinyl guaiacol, guaiacol, and protocatechuic acid) were also evaluated. The influence of medium composition,fermentation technology (batch or fed-batch), supplementation with vanillic acid, and inoculum concentration on ferulic acid biotransformation were evaluated. The results postulate the initial concentration of cell mass as the variable with the strongest impact on ferulic acid metabolization under the studied conditions. The highest amounts of vanillin and vanillic acid were achieved at intermediate values of cell mass.Vanillyl alcohol and protocatechuic acid were more closely linked to high cell mass concentrations. Conversely, 4-vinyl guaiacol reached its highest amount at the lowest amount of cell mass. Guaiacol was not detected in any case. Therefore,the initial cell concentration must be considered a critical parameter when using Amycolaptosis sp. ATCC 39116 for the production of vanillin and related compounds.

  1. Media optimization studies for Serratiopeptidase production from Serratia marcescens ATCC 13880.

    PubMed

    Badhe, Ravindra V; Nanda, Rabindra K; Kulkarni, Manasi B; Bhujbal, Mayur N; Patil, Pradeep S; Badhe, Sonali R

    2009-01-01

    Production of an anti-inflammatory enzyme serratiopeptidase by fermentation with Serratia marcescens ATCC 13880 was studied to ascertain optimal nutritional conditions for large scale production. To study biosynthesis and production of serratiopeptidase by Serratia marcescens ATCC 13880, different physicochemical parameters were studied and optimized. The optimized medium contain, (g/l) glycerine 10.0, maltose 10.0 as carbon source, peptone 10.0 as organic nitrogen source, ammonium sulphate 10.0 as inorganic nitrogen source, dihydrogen phosphate 10.0, sodium bicarbonate 10.0, sodium acetate 10.0 as inorganic salt source, ascorbic acid 10.0 as stabilizer, distilled water 1000 ml and the optimized fermentation conditions were pH 7.0, temperature 37 degrees C and duration 24 hr. The modified fermentation medium produced 27.36 IU/ml of serratiopeptidase compared to 17.97 IU/ml in basal medium and the molecular weight of the purified serratiopeptidase was found to be 52 kD.

  2. The Glycopeptide Antitumor Antibiotic Zorbamycin from Streptomyces flavoviridis ATCC 21892: Strain Improvement and Structure Elucidation⊥

    PubMed Central

    Wang, Liyan; Yun, Bong-Sik; George, Nicholas P.; Wendt-Pienkowski, Evelyn; Galm, Ute; Oh, Tae-Jin; Coughlin, Jane M.; Zhang, Guodong; Tao, Meifeng; Shen, Ben

    2008-01-01

    Zorbamycin (1, ZBM) is a glycopeptide antitumor antibiotic first reported in 1971. The partial structures of 1 were speculated on the basis of its acid hydrolysis products, but the structure of the intact molecule has never been established. The low titer of 1 from the wild-type strains, combined with its acid-instability, has so far hampered its isolation. By random mutagensis of Streptomyces flavoviridis ATCC21892, a wild-type producer of 1, with UV irradiation, two high-producing strains of 1, S. flavoviridis SB9000 and SB9001, were isolated. Under the optimized fermentation conditions, these two strains produced about 10 mg/L of 1, which was about 10-fold higher than the wild-type ATCC21892 strain, as estimated by HPLC analysis. Finally, 1 was isolated both as a 1-Cu complex and Cu-free molecule, and the intact structure of 1 was established on the basis of a combination of mass spectrometry and 1H and 13C NMR spectroscopic analyses. PMID:17311457

  3. Assessment of in vitro removal of cholesterol oxidation products by Lactobacillus casei ATCC334.

    PubMed

    Machorro-Méndez, I A; Hernández-Mendoza, A; Cardenia, V; Rodriguez-Estrada, M T; Lercker, G; Spinelli, F; Cellini, A; García, H S

    2013-11-01

    Cholesterol oxidation products (COPs) are a group of compounds formed during processing and storage of foods from animal origin. After ingestion, COPs are absorbed in the intestine and can be distributed to serum and various tissues, potentially promoting a variety of toxic effects. Therefore, inhibition of their intestinal absorption may contribute to reduce the health risks associated with dietary intake of COPs. Some studies have shown that drugs and dietary compounds may inhibit the intestinal absorption of dietary COPs. However, proven cholesterol- and/or food toxins-binding lactic acid bacteria have not been previously evaluated as potential COPs removal agents. The aim of this study was to assess the ability of Lactobacillus casei ATCC334 to remove COPs in aqueous solution. Results showed the ability of both growing and resting cells to remove COPs (ca. 30-60%). All COPs-bacterium interactions were specific and partly reversible, being resting cells the most efficient for COPs removal in a ranking order of 7-KC > 7α-OH/7β-OH > triol > 5,6β-EP > 5,6α-EP > 25-OH. Binding to the cell wall and/or cell membrane incorporation appears to be the most likely mechanisms involved on COPs removal by L. casei ATCC 334.

  4. An Evaluation of Kinetic Models in the Biodesulfurization of Synthetic Oil by Rhodococcus erythropolis ATCC 4277.

    PubMed

    Maass, D; Mayer, D A; Moritz, D E; Oliveira, D; de Souza, A A Ulson; Souza, S M A Guelli

    2015-10-01

    Biodesulfurization is an eco-friendly technology applied in the removal of sulfur from fossil fuels. This technology is based on the use of microorganisms as biocatalysts to convert the recalcitrant sulfur compounds into others easily treatable, as sulfides. Despite it has been studied during the last decades, there are some unsolved questions, as per example the kinetic model which appropriately describes the biodesulfurization globally. In this work, different kinetic models were tested to a batch desulfurization process using dibenzothiophene (DBT) as a model compound, n-dodecane as organic solvent, and Rhodococcus erythropolis ATCC 4277 as biocatalyst. The models were solved by ODE45 function in the MATLAB. Monod model was capable to describe the biodesulfurization process predicting all experimental data with a very good fitting. The coefficients of determination achieved to organic phase concentrations of 20, 80, and 100 % (v/v) were 0.988, 0.995, and 0.990, respectively. R. erythropolis ATCC 4277 presented a good affinity with the substrate (DBT) since the coefficients of saturation obtained to reaction medium containing 20, 80, and 100 % (v/v) were 0.034, 0.07, and 0.116, respectively. This kinetic evaluation provides an improvement in the development of biodesulfurization technology because it showed that a simple model is capable to describe the throughout process.

  5. Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142

    NASA Technical Reports Server (NTRS)

    Schneegurt, M. A.; Sherman, D. M.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

    1997-01-01

    Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O2-sensitive N2 fixation from oxygenic photosynthesis. The energy and reducing power needed for N2 fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture.

  6. Isolation and Characterization of a Novel Virulent Phage of Lactobacillus casei ATCC 393.

    PubMed

    Zhang, Xi; Lan, Yu; Jiao, Wenchao; Li, Yijing; Tang, Lijie; Jiang, Yanping; Cui, Wen; Qiao, Xinyuan

    2015-12-01

    A new virulent phage (Lcb) of Lactobacillus casei ATCC 393 was isolated from Chinese sauerkraut. It was specific to L. casei ATCC 393. Electron micrograph revealed that it had an icosahedral head (60.2 ± 0.8 nm in diameter) and a long tail (251 ± 2.6 nm). It belonged to the Siphoviridae family. The genome of phage Lcb was estimated to be approximately 40 kb and did not contain cohesive ends. One-step growth kinetics of its lytic development revealed latent and burst periods of 75 and 45 min, respectively, with a burst size of 16 PFU per infected cell. The phage was able to survive in a pH range between 4 and 11. However, a treatment of 70 °C for 30 min and 75% ethanol or isopropanol for 20 min was observed to inactivate phage Lcb thoroughly. The presence of both Ca(2+) and Mg(2+) showed a little influence on phage adsorption, but they were indispensable to gain complete lysis and improve plaque formation. The adsorption kinetics were similar on viable or nonviable cells, and high adsorption rates maintained between 10 and 37 °C. The highest adsorption rate was at 30 °C. This study increased the knowledge on phages of L. casei. The characterization of phage Lcb is helpful to establish a basis for adopting effective strategies to control phage attack in industry. PMID:26123178

  7. Lignan formation in hairy root cultures of Edelweiss (Leontopodium nivale ssp. alpinum (Cass.) Greuter)

    PubMed Central

    Wawrosch, Christoph; Schwaiger, Stefan; Stuppner, Hermann; Kopp, Brigitte

    2014-01-01

    A hairy root line of Edelweiss (Leontopodium nivale ssp. alpinum (Cass.) Greuter) was obtained upon transformation with Agrobacterium rhizogenes strain ATCC15834. Elicitation of this line with silver nitrate, sucrose, methyl jasmonate and yeast extract at various concentrations in most cases resulted in a stimulation of lignan biosynthesis. Through elicitation with 6% sucrose the roots accumulated the pharmacologically active lignans leoligin and 5-methoxy-leoligin at levels of 0.0678% and 0.0372%, respectively, without significant growth inhibition. These lignan levels were comparable to those found in intact roots of cultivated Edelweiss. The biotechnological production of leoligin could be an attractive option for the continuous, field culture-independent production of the valuable secondary metabolites leoligin and 5-methoxy-leoligin. PMID:24932777

  8. Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends.

    PubMed

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood

    2012-07-20

    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide. PMID:22582388

  9. Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions.

    PubMed

    Hwang, Hau-Hsuan; Liu, Yin-Tzu; Huang, Si-Chi; Tung, Chin-Yi; Huang, Fan-Chen; Tsai, Yun-Long; Cheng, Tun-Fang; Lai, Erh-Min

    2015-02-01

    Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an α-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth. PMID:25163013

  10. IMPa-4, an Arabidopsis importin alpha isoform, is preferentially involved in agrobacterium-mediated plant transformation.

    PubMed

    Bhattacharjee, Saikat; Lee, Lan-Ying; Oltmanns, Heiko; Cao, Hongbin; Veena; Cuperus, Joshua; Gelvin, Stanton B

    2008-10-01

    Successful transformation of plants by Agrobacterium tumefaciens requires that the bacterial T-complex actively escorts T-DNA into the host's nucleus. VirD2 and VirE2 are virulence proteins on the T-complex that have plant-functional nuclear localization signal sequences that may recruit importin alpha proteins of the plant for nuclear import. In this study, we evaluated the involvement of seven of the nine members of the Arabidopsis thaliana importin alpha family in Agrobacterium transformation. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that all tested Arabidopsis importin alpha members can interact with VirD2 and VirE2. However, only disruption of the importin IMPa-4 inhibited transformation and produced the rat (resistant to Agrobacterium transformation) phenotype. Overexpression of six importin alpha members, including IMPa-4, rescued the rat phenotype in the impa-4 mutant background. Roots of wild-type and impa-4 Arabidopsis plants expressing yellow fluorescent protein-VirD2 displayed nuclear localization of the fusion protein, indicating that nuclear import of VirD2 is not affected in the impa-4 mutant. Somewhat surprisingly, VirE2-yellow fluorescent protein mainly localized to the cytoplasm of both wild-type and impa-4 Arabidopsis cells and to the cytoplasm of wild-type tobacco (Nicotiana tabacum) cells. However, bimolecular fluorescence complementation assays indicated that VirE2 could localize to the nucleus when IMPa-4, but not when IMPa-1, was overexpressed. PMID:18836040

  11. Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends

    SciTech Connect

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood

    2012-09-05

    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

  12. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    PubMed

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

  13. Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions.

    PubMed

    Hwang, Hau-Hsuan; Liu, Yin-Tzu; Huang, Si-Chi; Tung, Chin-Yi; Huang, Fan-Chen; Tsai, Yun-Long; Cheng, Tun-Fang; Lai, Erh-Min

    2015-02-01

    Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an α-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth.

  14. High efficiency transformation ofBrassica napus usingAgrobacterium vectors.

    PubMed

    Moloney, M M; Walker, J M; Sharma, K K

    1989-04-01

    An efficient procedure for obtaining transgenicBrassica napus plants usingAgrobacterium binary vectors is described. The target tissue for the transformation is the cut end of cotyledonary petioles. These tissues, when cultured with their lamina intact, show a regeneration frequency of more than 80%. The cells of this cut surface, which undergo organogenesis, are very susceptible to topical infection byAgrobacterium. The cocultivation method used does not require feeder layers or use of exogenously applied promoters of virulence. After 72h of infection withAgrobacterium the explants were transferred to selective regeneration medium. Using kanamycin (15μg cm(-3)) for selection, transgenic plantlets emerged within 3 weeks. These plantlets which appeared on over half the explants were excised and rooted for a further 7-10 days. When the plants were large enough, leaves were taken for assay of NPT II activity using dot blots. Most of the plants surviving the selection showed substantial NPT II activity. The frequency of transformation and yield of transgenic plants was higher than in previously reported methods with this species. Southern blotting revealed that integration of the T-DNA frequently occurred in multiple copies and at multiple loci in the genome. The transgenicB. napus plants all grew normally and developed fertile flowers. The transgenic plants were self-pollinated and their progeny studied by two methods. The first was a single-embryo NPT II assay performed on developing seeds of these selfed-plants. The second was a leaf bleaching assay performed by selection of germinating seedlings of the selfed progeny. Both assays yielded segregation ratios consistent with the number of integration events indicated by Southern blots. The method should have broad application in studies of gene expression in theBrassicaceae and will be a cost-effective alternative to those seeking to improveBrassica crops by introduction of foreign genes.

  15. Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

    PubMed

    Buschmann, Henrik

    2016-01-01

    The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

  16. Direct visualization of Agrobacterium-delivered VirE2 in recipient cells

    PubMed Central

    Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q

    2014-01-01

    Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 μm sec−1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048

  17. Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.

    PubMed

    Li, Xiaoyang; Yang, Qinghua; Tu, Haitao; Lim, Zijie; Pan, Shen Q

    2014-02-01

    Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T-DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1-10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3-3.1 μm sec⁻¹ in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells. PMID:24299048

  18. VirB/D4-dependent protein translocation from Agrobacterium into plant cells.

    PubMed

    Vergunst, A C; Schrammeijer, B; den Dulk-Ras, A; de Vlaam, C M; Regensburg-Tuïnk, T J; Hooykaas, P J

    2000-11-01

    The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease. In addition, several Virulence proteins must somehow be transported to fulfill a function in planta. Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells. Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA. PMID:11062129

  19. Agrobacterium-mediated inoculation of chrysanthemum (Chrysanthemum morifolium) plants with chrysanthemum stunt viroid.

    PubMed

    Nabeshima, Tomoyuki; Doi, Motoaki; Hosokawa, Munetaka

    2016-08-01

    Agroinfiltration was tested as a method of inoculation of chrysanthemum plants with chrysanthemum stunt viroid (CSVd). Binary vectors harboring dimeric CSVd sequences in sense and antisense orientations were constructed, and Agrobacterium transfected with these binary vectors was infiltrated into chrysanthemum leaves. Northern blotting and reverse transcription polymerase chain reaction analysis showed that local infection was established within 7 days and systemic infection within 20 days. CSVd polarities showed no difference in infectivity. This study showed that agroinfiltration of chrysanthemum plants is an easy, rapid, and cost-effective method for CSVd inoculation. PMID:27155239

  20. Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58.

    PubMed

    Goodner, B; Hinkle, G; Gattung, S; Miller, N; Blanchard, M; Qurollo, B; Goldman, B S; Cao, Y; Askenazi, M; Halling, C; Mullin, L; Houmiel, K; Gordon, J; Vaudin, M; Iartchouk, O; Epp, A; Liu, F; Wollam, C; Allinger, M; Doughty, D; Scott, C; Lappas, C; Markelz, B; Flanagan, C; Crowell, C; Gurson, J; Lomo, C; Sear, C; Strub, G; Cielo, C; Slater, S

    2001-12-14

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

  1. Cellulitis and myositis caused by Agrobacterium radiobacter and Haemophilus parainfluenzae after influenza virus vaccination.

    PubMed

    Owensby, J E; Elliott, S; Tu, K; Hernandez, J E

    1997-07-01

    Agrobacterium radiobacter is a gram-negative aerobic bacillus that has been reported as a cause of disease only 36 times in the literature. More than half of the patients (25) have had bacteremia. Peritonitis, urinary tract infection, endocarditis, and one case of cellulitis associated with bacteremia have also been reported. Infection is often associated with immunosuppression and the presence of a plastic foreign body, such as central venous catheters, nephrostomy tubes, intraperitoneal catheters, and prosthetic cardiac valves. We present apparently the first case of A radiobacter causing myositis after influenza virus vaccination.

  2. [Agrobacterium tumefaciens (Radiobacter) as an infectious agent in an oncological patient].

    PubMed

    Franková, H; Churý, Z; Gaislerová, V; Jílková, J; Hejlová, N; Mach, J

    1999-05-01

    The authors submit the description of a 62-year-old patient with multiple myeloma where the causal agent of pyretic reactions was Agrobacterium tumefaciens (radiobacter). It was a patient with an implanted venous port which was colonized by the above bacterium. This finding most probably has not been described so far in the Czech literature. In the English literature the authors found 36 cases. The authors draw attention to the possible higher incidence of future infections caused by organisms hitherto considered non-pathogenic for man, in particular in immunocompromised patients.

  3. Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation

    NASA Astrophysics Data System (ADS)

    Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina

    This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.

  4. Agrobacterium-mediated transformation of tomato with the ICE1 transcription factor gene.

    PubMed

    Juan, J X; Yu, X H; Jiang, X M; Gao, Z; Zhang, Y; Li, W; Duan, Y D; Yang, G

    2015-01-30

    ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower.

  5. Transgenic sugar beet tolerant to imidazolinone obtained by Agrobacterium-mediated transformation.

    PubMed

    Kishchenko, E M; Komarnitskii, I K; Kuchuk, N V

    2011-01-01

    Sugar beet is highly sensitive to imidazolinone herbicides thus rotational restrictions exist. In order to develop imidazolinone tolerant sugar beets als gene from Arabidopsis thaliana encoding acetolactate synthase with S653N mutation was used for genetic transformation. Transgenic sugar beet plants were obtained by Agrobacterium-mediated transformation of aseptic seedlings using vacuum-infiltration. The efficiency of genetic transformation was 5.8%. RT-PCR analysis of obtained plants revealed accumulation of specific als transcript. The resistance to imidazolinone was proved for developed transgenic sugar beet plants in vitro and in greenhouse conditions after spraying with imazethapyr (Pursuit, BASF).

  6. Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants.

    PubMed

    McKently, A H; Moore, G A; Doostdar, H; Niedz, R P

    1995-08-01

    Transgenic peanut (Arachis hypogaea L.) plants have been produced using an Agrobacterium-mediated transformation system. Zygotic embryo axes from mature seed were cocultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector that contained the genes for the scorable marker B-glucuronidase (GUS) and the selectable marker neomycin phosphotransferase II. Nine percent of the germinated seedlings were GUS+. Polymerase chain reaction analysis confirmed that GUS+ shoots and T1 progeny contained T-DNA. Molecular characterization of one primary transformant and its T1 and T2 progeny plants established that T-DNA was integrated into the host genome. PMID:24186625

  7. Catheter-related bacteremia caused by Agrobacterium radiobacter in a cancer patient: case report and literature review.

    PubMed

    Paphitou, N I; Rolston, K V I

    2003-12-01

    Agrobacteria are a group of phytopathogenic organisms widely distributed in soil; they are now recognized as rare human pathogens affecting mostly immunocompromised hosts. We report a case of catheter-related bacteremia due to Agrobacterium radiobacter in a neutropenic patient and describe the clinical presentations, treatment strategies and outcome of Agrobacterium infections based on our experience and a literature review. The antimicrobial susceptibility patterns of these organisms appear to be quite variable and collective susceptibility data derived from this and previous reports are provided.

  8. Complete genome sequence of Actinobacillus equuli subspecies equuli ATCC 19392T

    PubMed Central

    2015-01-01

    Actinobacillus equuli subsp. equuli is a member of the family Pasteurellaceae that is a common resident of the oral cavity and alimentary tract of healthy horses. At the same time, it can also cause a fatal septicemia in foals, commonly known as sleepy foal disease or joint ill disease. In addition, A. equuli subsp. equuli has recently been reported to act as a primary pathogen in breeding sows and piglets. To better understand how A. equuli subsp. equuli can cause disease, the genome of the type strain of A. equuli subsp. equuli, ATCC 19392T, was sequenced using the PacBio RSII sequencing system. Its genome is comprised of 2,431,533 bp and is predicted to encode 2,264 proteins and 82 RNAs. PMID:26203343

  9. Response of electrically stimulated cells of Pseudomonas oleovorans strain ATCC 29347 suspended in silicone oil.

    PubMed

    Anglade, J; Hirschler, A; Le Petit, J; Matheron, R; Scarpitta, A; Iacazio, G

    2001-05-15

    A high intensity direct current was applied for more than 10 min onto a bacterial suspension of Pseudomonas oleovorans ATCC 29347 suspended in silicone oil. The application of a gradually increased electric field from 0 to 2500 V x cm(-1) resulted in a decrease of the optical density of the bacterial suspension and the occurrence of a peak current of several hundred microA for living cells instead of a linear increase (few microA) for killed or lyophilised cells. This procedure is not only a rapid way of investigating the living state of cell cultures but also an efficient experimental tool to study the cellular effects of a controlled electrical stress. PMID:11356578

  10. Direct observation of redox reactions in Candida parapsilosis ATCC 7330 by Confocal microscopic studies

    PubMed Central

    Venkataraman, Sowmyalakshmi; Narayan, Shoba; Chadha, Anju

    2016-01-01

    Confocal microscopic studies with the resting cells of yeast, Candida parapsilosis ATCC 7330, a reportedly versatile biocatalyst for redox enzyme mediated preparation of optically pure secondary alcohols in high optical purities [enantiomeric excess (ee) up to >99%] and yields, revealed that the yeast cells had large vacuoles under the experimental conditions studied where the redox reaction takes place. A novel fluorescence method was developed using 1-(6-methoxynaphthalen-2-yl)ethanol to track the site of biotransformation within the cells. This alcohol, itself non-fluorescent, gets oxidized to produce a fluorescent ketone, 1-(6-methoxynaphthalen-2-yl)ethanone. Kinetic studies showed that the reaction occurs spontaneously and the products get released out of the cells in less time [5 mins]. The biotransformation was validated using HPLC. PMID:27739423

  11. Closing the Carbon Balance for Fermentation by Clostridium thermocellum (ATCC 27405)

    SciTech Connect

    Ellis, Lucas D; Holwerda, Evert K; Hogsett, David; Rogers, Steve; Shao, Xiongjun; Tschaplinski, Timothy J; Thorne, Phil; Lynd, L.

    2012-01-01

    Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, {>=}92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products - ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively.

  12. Pore-forming ability of major outer membrane proteins from Wolinella recta ATCC 33238.

    PubMed Central

    Kennell, W L; Egli, C; Hancock, R E; Holt, S C

    1992-01-01

    Three major outer membrane proteins with apparent molecular masses of 43, 45, and 51 kDa were purified from Wolinella recta ATCC 33238, and their pore-forming abilities were determined by the black lipid bilayer method. The non-heat-modifiable 45-kDa protein (Omp 45) showed no pore-forming activity even at high KCl concentrations. The single-channel conductances in 1 M KCl of the heat-modifiable proteins with apparent molecular masses of 43 kDa (Omp 43) and 51 kDa (Omp 51) were 0.49 and 0.60 nS, respectively. The proteins formed nonselective channels and, as determined by experiments of ion selectivity and zero-current potential, were weakly anion selective. Images PMID:1370429

  13. L-serine enhances the anaerobic lactate metabolism of Veillonella dispar ATCC 17745.

    PubMed

    Hoshino, E

    1987-06-01

    Under anaerobic conditions, the rate of metabolism of lactate by starved resting cells of Veillonella dispar ATCC 17745 was very low. Because pyruvate was metabolized well by the starved cells, oxidation of lactate to pyruvate, which is the first step of the lactate metabolism, must have been limited in the cells. In the starved cells, the levels of the metabolic intermediates, oxalacetate or fumarate, of which reductions to malate or to succinate could be coupled with lactate oxidation to pyruvate and initiate lactate metabolism, were quite low, suggesting that these had been reduced during the starvation steps under strictly anaerobic conditions. Thus, the starved cells were unable to start the anaerobic lactate metabolism because of shortage of such reducible substrates. L-serine greatly enhanced anaerobic lactate metabolism of the starved cells. This enhancement may have been due to metabolism of L-serine itself and conversion to oxalacetate and fumarate, which made it possible to begin lactate oxidation.

  14. Ala(0)-actagardine, a new lantibiotic from cultures of Actinoplanes liguriae ATCC 31048.

    PubMed

    Vértesy, L; Aretz, W; Bonnefoy, A; Ehlers, E; Kurz, M; Markus, A; Schiell, M; Vogel, M; Wink, J; Kogler, H

    1999-08-01

    The actagardine-producing strain Actinoplanes liguriae ATCC 31048, forms an additional lantibiotic when it is cultured on mannitol and soya meal. The new compound, Ala(0)-actagardine (1), has been isolated by solid-phase extraction followed by a two-step chromatographic separation. The molecular formula of 1 is C84H129N21O25S4. Its chemical structure was determined by 2D-NMR analysis and was further confirmed by an amino acid analysis, Edman degradation, and partial synthesis from actagardine. 1 exhibits a slightly higher biological activity than the parent compound actagardine. The synthetic analogs Lys(0)-actagardine (2) and Ile(0)-actagardine (3) demonstrate also antibacterial activities and emphasize the importance of the N-terminus for further derivatization. PMID:10580386

  15. Microbial conversion of ethylbenzene to 1-phenethanol and acetophenone by Nocardia tartaricans ATCC 31190.

    PubMed

    Cox, D P; Goldsmith, C D

    1979-09-01

    A culture of Nocardia tartaricans ATCC 31190 was capable of catalyzing the conversion of ethylbenzene to 1-phenethanol and acetophenone while growing in a shake flask culture with hexadecane as the source of carbon and energy. This subterminal oxidative reaction with ethylbenzene appears not to have been previously reported for Nocardia species. When N. tartaricans was grown on glucose as its source of carbon and energy and ethylbenzene was added, no subsequent production of 1-phenethanol or acetophenone was observed. The mechanisms of 1-phenethanol and acetophenone production from ethylbenzene are thought to involve a subterminal oxidation of the alpha-carbon of the alkyl group to 1-phenethanol followed by biological oxidation of the latter to acetophenone.

  16. Pullulan Production by Aureobasidium pullulans ATCC 201253 Cells Adsorbed onto Cellulose Anion and Cation Exchangers

    PubMed Central

    West, Thomas P.

    2012-01-01

    The anion exchanger phosphocellulose and the cation exchanger triethylaminoethyl cellulose were used to immobilize cells of the fungus Aureobasidium pullulans ATCC 201253 and the adsorbed cells were subsequently investigated for their ability to produce the polysaccharide pullulan using batch fermentation. The cells adsorbed on the triethylaminoethyl cellulose at pH 7.5 produced higher pullulan levels than those cells immobilized on phosphocellulose at pH 4.0 for 2 cycles of 168 h at 30 °C. Relative to the initial cycle of 168 h, pullulan production by the cells immobilized on the triethylaminoethyl cellulose decreased slightly after 168 h of the second production cycle while pullulan production by the phosphocellulose-immobilized cells remained about the same after 168 h of the second production cycle. PMID:23762749

  17. Effects of Salt Stress on Carbohydrate Metabolism of Lactobacillus plantarum ATCC 14917.

    PubMed

    Wang, Pingping; Wu, Zhen; Wu, Jing; Pan, Daodong; Zeng, Xiaoqun; Cheng, Kemeng

    2016-10-01

    Lactic acid bacteria are widely used in fermented foods, especially cheese products. In this study, we observed the salt tolerance of Lactobacillus plantarum ATCC 14917 after exposure to different concentrations of NaCl in MRS medium. Quantitative proteomic profiles using two-dimensional electrophoresis identified 384 proteins, of which 26 were upregulated and 31 downregulated. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry was then used to identify 11 proteins, of which three were linked to carbohydrate metabolism. The downregulation of carbamoyl phosphate synthase in carbohydrate metabolism revealed a bacterial regulation mechanism to save energy in order to survive during the salt tolerance. Other proteins were found involved in transcription-translation processes, fatty acid biosynthesis, and the primary metabolic process.

  18. Biotransformation of the Antimelanoma Agent Betulinic Acid by Bacillus megaterium ATCC 13368

    PubMed Central

    Chatterjee, Parnali; Kouzi, Samir A.; Pezzuto, John M.; Hamann, Mark T.

    2000-01-01

    Microbial transformation of the antimelanoma agent betulinic acid was studied. The main objective of this study was to utilize microorganisms as in vitro models to predict and prepare potential mammalian metabolites of this compound. Preparative-scale biotransformation with resting-cell suspensions of Bacillus megaterium ATCC 13368 resulted in the production of four metabolites, which were identified as 3-oxo-lup-20(29)-en-28-oic acid, 3-oxo-11α-hydroxy-lup-20(29)-en-28-oic acid, 1β-hydroxy-3-oxo-lup-20(29)-en-28-oic acid, and 3β,7β,15α-trihydroxy-lup-20(29)-en-28-oic acid based on nuclear magnetic resonance and high-resolution mass spectral analyses. In addition, the antimelanoma activities of these metabolites were evaluated with two human melanoma cell lines, Mel-1 (lymph node) and Mel-2 (pleural fluid). PMID:10966400

  19. Resistance to vanadium in Pseudomonas fluorescens ATCC 17400 caused by mutations in TCA cycle enzymes.

    PubMed

    Denayer, Sarah; Matthijs, Sandra; Cornelis, Pierre

    2006-11-01

    Vanadium inhibits the growth of Pseudomonas fluorescens ATCC 17400 in the low-iron casamino acids medium and even more when iron is added to the medium. Analysis of transposon mutants allowed the isolation of two mutants with increased resistance to vanadium. One mutant had an insertion in the idh gene coding for the tricarboxylic acid enzyme isocitrate dehydrogenase. The second mutant had the transposon inserted into acnD, one out of three genes coding for a 2-methyl-isocitrate dehydratase (aconitase). In this mutant, there was a higher level of acnB aconitase transcripts while the levels of acnA transcripts were unchanged. A nonpolar idh mutant was obtained, which showed the same level of resistance against vanadium as the original transposon mutant. PMID:17020548

  20. Effects of Salt Stress on Carbohydrate Metabolism of Lactobacillus plantarum ATCC 14917.

    PubMed

    Wang, Pingping; Wu, Zhen; Wu, Jing; Pan, Daodong; Zeng, Xiaoqun; Cheng, Kemeng

    2016-10-01

    Lactic acid bacteria are widely used in fermented foods, especially cheese products. In this study, we observed the salt tolerance of Lactobacillus plantarum ATCC 14917 after exposure to different concentrations of NaCl in MRS medium. Quantitative proteomic profiles using two-dimensional electrophoresis identified 384 proteins, of which 26 were upregulated and 31 downregulated. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry was then used to identify 11 proteins, of which three were linked to carbohydrate metabolism. The downregulation of carbamoyl phosphate synthase in carbohydrate metabolism revealed a bacterial regulation mechanism to save energy in order to survive during the salt tolerance. Other proteins were found involved in transcription-translation processes, fatty acid biosynthesis, and the primary metabolic process. PMID:27342422

  1. A partial proteome reference map of the wine lactic acid bacterium Oenococcus oeni ATCC BAA-1163.

    PubMed

    Mohedano, María de la Luz; Russo, Pasquale; de Los Ríos, Vivian; Capozzi, Vittorio; Fernández de Palencia, Pilar; Spano, Giuseppe; López, Paloma

    2014-02-26

    Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

  2. Complete genome sequence of Streptomyces venezuelae ATCC 15439, a promising cell factory for production of secondary metabolites.

    PubMed

    Song, Ju Yeon; Yoo, Young Ji; Lim, Si-Kyu; Cha, Sun Ho; Kim, Ji-Eun; Roe, Jung-Hye; Kim, Jihyun F; Yoon, Yeo Joon

    2016-02-10

    Streptomyces venezuelae ATCC 15439, which produces 12- and 14-membered ring macrolide antibiotics, is a platform strain for heterologous expression of secondary metabolites. Its 9.05-Mb genome sequence revealed an abundance of genes involved in the biosynthesis of secondary metabolites and their precursors, which should be useful for the production of bioactive compounds. PMID:26718561

  3. Complete genome sequence of Streptomyces venezuelae ATCC 15439, a promising cell factory for production of secondary metabolites.

    PubMed

    Song, Ju Yeon; Yoo, Young Ji; Lim, Si-Kyu; Cha, Sun Ho; Kim, Ji-Eun; Roe, Jung-Hye; Kim, Jihyun F; Yoon, Yeo Joon

    2016-02-10

    Streptomyces venezuelae ATCC 15439, which produces 12- and 14-membered ring macrolide antibiotics, is a platform strain for heterologous expression of secondary metabolites. Its 9.05-Mb genome sequence revealed an abundance of genes involved in the biosynthesis of secondary metabolites and their precursors, which should be useful for the production of bioactive compounds.

  4. Genome Sequence of Streptococcus phocae subsp. phocae Strain ATCC 51973T Isolated from a Harbor Seal (Phoca vitulina)

    PubMed Central

    Poblete-Morales, Matías

    2015-01-01

    Streptococcus phocae subsp. phocae is a pathogen that affects different pinniped and mammalian species. This announcement reports the genome sequence of the type strain ATCC 51973 isolated in Norway from clinical specimens of harbor seal (Phoca vitulina), revealing interesting genes related to possible virulence factors. PMID:26586875

  5. Effect of Lactobacillus brevis ATCC 8287 as a feeding supplement on the performance and immune function of piglets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lactobacillus brevis ATCC 8287, a surface (S-layer) strain, possesses a variety of functional properties that make it both a potential probiotic and a good vaccine vector candidate. With this in mind, our aim was to study the survival of L. brevis in the porcine gut and investigate the effect of th...

  6. Complete Genome Sequence of Gluconacetobacter hansenii Strain NQ5 (ATCC 53582), an Efficient Producer of Bacterial Cellulose.

    PubMed

    Pfeffer, Sarah; Mehta, Kalpa; Brown, R Malcolm

    2016-01-01

    This study reports the release of the complete nucleotide sequence of Gluconacetobacter hansenii strain NQ5 (ATCC 53582). This strain was isolated by R. Malcolm Brown, Jr. in a sugar mill in North Queensland, Australia, and is an efficient producer of bacterial cellulose. The elucidation of the genome will contribute to the study of the molecular mechanisms necessary for cellulose biosynthesis. PMID:27516505

  7. Genome sequence alterations detected upon passage of Burkholderia mallei ATCC 23344 in culture and in mammalian hosts

    PubMed Central

    Romero, Claudia M; DeShazer, David; Feldblyum, Tamara; Ravel, Jacques; Woods, Donald; Kim, H Stanley; Yu, Yan; Ronning, Catherine M; Nierman, William C

    2006-01-01

    Background More than 12,000 simple sequence repeats (SSRs) have been identified in the genome of Burkholderia mallei ATCC 23344. As a demonstrated mechanism of phase variation in other pathogenic bacteria, these may function as mutable loci leading to altered protein expression or structure variation. To determine if such alterations are occurring in vivo, the genomes of various single-colony passaged B. mallei ATCC 23344 isolates, one from each source, were sequenced from culture, a mouse, a horse, and two isolates from a single human patient, and the sequence compared to the published B. mallei ATCC 23344 genome sequence. Results Forty-nine insertions and deletions (indels) were detected at SSRs in the five passaged strains, a majority of which (67.3%) were located within noncoding areas, suggesting that such regions are more tolerant of sequence alterations. Expression profiling of the two human passaged isolates compared to the strain before passage revealed alterations in the mRNA levels of multiple genes when grown in culture. Conclusion These data support the notion that genome variability upon passage is a feature of B. mallei ATCC23344, and that within a host B. mallei generates a diverse population of clones that accumulate genome sequence variation at SSR and other loci. PMID:16953889

  8. Complete Genome Sequence of Gluconacetobacter hansenii Strain NQ5 (ATCC 53582), an Efficient Producer of Bacterial Cellulose.

    PubMed

    Pfeffer, Sarah; Mehta, Kalpa; Brown, R Malcolm

    2016-08-11

    This study reports the release of the complete nucleotide sequence of Gluconacetobacter hansenii strain NQ5 (ATCC 53582). This strain was isolated by R. Malcolm Brown, Jr. in a sugar mill in North Queensland, Australia, and is an efficient producer of bacterial cellulose. The elucidation of the genome will contribute to the study of the molecular mechanisms necessary for cellulose biosynthesis.

  9. Complete Genome Sequence of Gluconacetobacter hansenii Strain NQ5 (ATCC 53582), an Efficient Producer of Bacterial Cellulose

    PubMed Central

    Pfeffer, Sarah; Mehta, Kalpa

    2016-01-01

    This study reports the release of the complete nucleotide sequence of Gluconacetobacter hansenii strain NQ5 (ATCC 53582). This strain was isolated by R. Malcolm Brown, Jr. in a sugar mill in North Queensland, Australia, and is an efficient producer of bacterial cellulose. The elucidation of the genome will contribute to the study of the molecular mechanisms necessary for cellulose biosynthesis. PMID:27516505

  10. Meta-analysis: Lactobacillus reuteri strain DSM 17938 (and the original strain ATCC 55730) for treating acute gastroenteritis in children.

    PubMed

    Szajewska, H; Urbańska, M; Chmielewska, A; Weizman, Z; Shamir, R

    2014-09-01

    Lactobacillus reuteri ATCC 55730 has been shown to provide a moderate clinical effect in the treatment of acute gastroenteritis (AGE) in children. However, as the L. reuteri ATCC 55730 strain was found to carry potentially transferable resistance traits for tetracycline and lincomycin, it was replaced by a new strain, L. reuteri DSM 17938, without unwanted plasmid-borne antibiotic resistance. Bioequivalence of the two strains has been suggested. We aimed to systematically evaluate data on the effectiveness of L. reuteri DSM 17938 and the original strain, L. reuteri ATCC 55730, in the treatment of AGE in children. The Cochrane Library, MEDLINE, and EMBASE databases, reference lists, and abstract books of major scientific meetings were searched in August 2013, with no language restrictions, for relevant randomised controlled trials (RCTs). Two RCTs (n=196) that evaluated L. reuteri DSM 17938 and three RCTs (n=156) that evaluated L. reuteri ATCC 55730, which involved hospitalised children aged 3 to 60 months, met the inclusion criteria. Compared with placebo or no treatment, DSM 17938 significantly reduced the duration of diarrhoea (mean difference -32 h, 95% confidence interval (CI): -41 to -24) and increased the chance of cure on day 3 (relative risk: 3.5, 95% CI: 1.2 to 10.8, random effects model). Similar results were obtained with the original strain, L. reuteri ATCC 55730. In conclusion, in hospitalised children, use of both strains of L. reuteri reduced the duration of diarrhoea, and more children were cured within 3 days. Data from outpatients and countryspecific cost-effectiveness analyses are needed. Given the limited data and the methodological limitations of the included trials, the evidence should be viewed with caution.

  11. In situ hydrogen, acetone, butanol, ethanol and microdiesel production by Clostridium acetobutylicum ATCC 824 from oleaginous fungal biomass.

    PubMed

    Hassan, Elhagag Ahmed; Abd-Alla, Mohamed Hemida; Bagy, Magdy Mohamed Khalil; Morsy, Fatthy Mohamed

    2015-08-01

    An in situ batch fermentation technique was employed for biohydrogen, acetone, butanol, ethanol and microdiesel production from oleaginous fungal biomass using the anaerobic fermentative bacterium Clostridium acetobutylicum ATCC 824. Oleaginous fungal Cunninghamella echinulata biomass which has ability to accumulate up to 71% cellular lipid was used as the substrate carbon source. The maximum cumulative hydrogen by C. acetobutylicum ATCC 824 from crude C. echinulata biomass was 260 ml H2 l(-1), hydrogen production efficiency was 0.32 mol H2 mole(-1) glucose and the hydrogen production rate was 5.2 ml H2 h(-1). Subsequently, the produced acids (acetic and butyric acids) during acidogenesis phase are re-utilized by ABE-producing clostridia and converted into acetone, butanol, and ethanol. The total ABE produced by C. acetobutylicum ATCC 824 during batch fermentation was 3.6 g l(-1) from crude fungal biomass including acetone (1.05 g l(-1)), butanol (2.19 g l(-1)) and ethanol (0.36 g l(-1)). C. acetobutylicum ATCC 824 has ability to produce lipolytic enzymes with a specific activity 5.59 U/mg protein to hydrolyze ester containing substrates. The lipolytic potential of C. acetobutylicum ATCC 824 was used as a biocatalyst for a lipase transesterification process using the produced ethanol from ABE fermentation for microdiesel production. The fatty acid ethyl esters (microdiesel) generated from the lipase transesterification of crude C. echinulata dry mass was analyzed by GC/MS as 15.4% of total FAEEs. The gross energy content of biohydrogen, acetone, butanol, ethanol and biodiesel generated through C. acetobutylicum fermentation from crude C. echinulata dry mass was 3113.14 kJ mol(-1). These results suggest a possibility of integrating biohydrogen, acetone, butanol and ethanol production technology by C. acetobutylicum with microdiesel production from crude C. echinulata dry mass and therefore improve the feasibility and commercialization of bioenergy production.

  12. Investigation of the Amycolatopsis sp. Strain ATCC 39116 Vanillin Dehydrogenase and Its Impact on the Biotechnical Production of Vanillin

    PubMed Central

    Fleige, Christian; Hansen, Gunda; Kroll, Jens

    2013-01-01

    The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDHATCC 39116). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDHATCC 39116 was purified to apparent electrophoretic homogeneity and exhibited NAD+-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Kmr mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Kmr mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin. PMID:23064333

  13. In situ hydrogen, acetone, butanol, ethanol and microdiesel production by Clostridium acetobutylicum ATCC 824 from oleaginous fungal biomass.

    PubMed

    Hassan, Elhagag Ahmed; Abd-Alla, Mohamed Hemida; Bagy, Magdy Mohamed Khalil; Morsy, Fatthy Mohamed

    2015-08-01

    An in situ batch fermentation technique was employed for biohydrogen, acetone, butanol, ethanol and microdiesel production from oleaginous fungal biomass using the anaerobic fermentative bacterium Clostridium acetobutylicum ATCC 824. Oleaginous fungal Cunninghamella echinulata biomass which has ability to accumulate up to 71% cellular lipid was used as the substrate carbon source. The maximum cumulative hydrogen by C. acetobutylicum ATCC 824 from crude C. echinulata biomass was 260 ml H2 l(-1), hydrogen production efficiency was 0.32 mol H2 mole(-1) glucose and the hydrogen production rate was 5.2 ml H2 h(-1). Subsequently, the produced acids (acetic and butyric acids) during acidogenesis phase are re-utilized by ABE-producing clostridia and converted into acetone, butanol, and ethanol. The total ABE produced by C. acetobutylicum ATCC 824 during batch fermentation was 3.6 g l(-1) from crude fungal biomass including acetone (1.05 g l(-1)), butanol (2.19 g l(-1)) and ethanol (0.36 g l(-1)). C. acetobutylicum ATCC 824 has ability to produce lipolytic enzymes with a specific activity 5.59 U/mg protein to hydrolyze ester containing substrates. The lipolytic potential of C. acetobutylicum ATCC 824 was used as a biocatalyst for a lipase transesterification process using the produced ethanol from ABE fermentation for microdiesel production. The fatty acid ethyl esters (microdiesel) generated from the lipase transesterification of crude C. echinulata dry mass was analyzed by GC/MS as 15.4% of total FAEEs. The gross energy content of biohydrogen, acetone, butanol, ethanol and biodiesel generated through C. acetobutylicum fermentation from crude C. echinulata dry mass was 3113.14 kJ mol(-1). These results suggest a possibility of integrating biohydrogen, acetone, butanol and ethanol production technology by C. acetobutylicum with microdiesel production from crude C. echinulata dry mass and therefore improve the feasibility and commercialization of bioenergy production. PMID

  14. An improved plant regeneration and Agrobacterium - mediated transformation of red pepper (Capsicum annuum L.).

    PubMed

    Kumar, R Vinoth; Sharma, V K; Chattopadhyay, B; Chakraborty, S

    2012-10-01

    Capsicum annuum (red pepper) is an important spice cum vegetable crop in tropical and subtropical countries. Here, we report an effective and reproducible auxin free regeneration method for six different red pepper cultivars (ACA-10, Kashi Anmol, LCA-235, PBC-535, Pusa Jwala and Supper) using hypocotyl explants and an efficient Agrobacterium-mediated transformation protocol. The explants (hypocotyls, cotyledonary leaves and leaf discs) collected from axenic seedlings of six red pepper cultivars were cultured on either hormone free MS medium or MS medium supplemented with BAP alone or in combination with IAA. Inclusion of IAA in the regeneration medium resulted in callus formation at the cut ends of explants, formation of rosette leaves and ill defined shoot buds. Regeneration of shoot buds could be achieved from hypocotyls grown in MS medium supplemented with different concentrations of BAP unlike other explants which failed to respond. Incorporation of GA3 in shoot elongation medium at 0.5 mg/l concentration enhanced the elongation in two cultivars, LCA-235 and Supper, while other cultivars showed no significant response. Chilli cultivar, Pusa Jwala was transformed with βC1 ORF of satellite DNA β molecule associated with Chilli leaf curl Joydebpur virus through Agrobacterium tumefaciens. Transgene integration in putative transformants was confirmed by PCR and Southern hybridization analysis.

  15. Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

    PubMed Central

    Ibrahim, Evra Raunie

    2014-01-01

    Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

  16. Stable Agrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection

    PubMed Central

    Alvarez, José M.; Ordás, Ricardo J.

    2013-01-01

    An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth. PMID:24376383

  17. Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability*

    PubMed Central

    Song, Zhang-yue; Tian, Jing-luan; Fu, Wei-zhe; Li, Lin; Lu, Ling-hong; Zhou, Lian; Shan, Zhi-hui; Tang, Gui-xiang; Shou, Hui-xia

    2013-01-01

    The Agrobacterium-mediated transformation system is the most commonly used method in soybean transformation. Screening of soybean genotypes favorable for Agrobacterium-infection and tissue regeneration is the most important step to establish an efficient genetic transformation system. In this study, twenty soybean genotypes that originated from different soybean production regions in China were screened for transient infection, regeneration capacity, and stable transgenic efficiency. Three genotypes, Yuechun 04-5, Yuechun 03-3, and Tianlong 1, showed comparable stable transgenic efficiencies with that of the previously reported American genotypes Williams 82 and Jack in our experimental system. For the Tianlong 1, the average stable transformation efficiency is 4.59%, higher than that of control genotypes (Jack and Williams 82), which is enough for further genomic research and genetic engineering. While polymerase chain reaction (PCR), LibertyLink strips, and β-glucuronidase (GUS) staining assays were used to detect the insertion and expression of the transgene, leaves painted with 135 mg/L Basta could efficiently identify the transformants. PMID:23549846

  18. Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration.

    PubMed

    van Attikum, H; Bundock, P; Hooykaas, P J

    2001-11-15

    Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T-DNA), derived from its tumour-inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T-DNA also to monocotyledonous plants, yeasts and fungi. The T-DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T-DNA recipient to demonstrate that the non-homologous end-joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome. We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements. PMID:11707425

  19. Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods.

    PubMed

    Joyce, Priya; Hermann, Scott; O'Connell, Anthony; Dinh, Quang; Shumbe, Leonard; Lakshmanan, Prakash

    2014-05-01

    Future genetic improvement of sugarcane depends, in part, on the ability to produce high-yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium-mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5', 3' or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques.

  20. Influence of volatile organic compounds emitted by Pseudomonas and Serratia strains on Agrobacterium tumefaciens biofilms.

    PubMed

    Plyuta, Vladimir; Lipasova, Valentina; Popova, Alexandra; Koksharova, Olga; Kuznetsov, Alexander; Szegedi, Erno; Chernin, Leonid; Khmel, Inessa

    2016-07-01

    The ability to form biofilms plays an important role in bacteria-host interactions, including plant pathogenicity. In this work, we investigated the action of volatile organic compounds (VOCs) produced by rhizospheric strains of Pseudomonas chlororaphis 449, Pseudomonas fluorescens B-4117, Serratia plymuthica IC1270, as well as Serratia proteamaculans strain 94, isolated from spoiled meat, on biofilms formation by three strains of Agrobacterium tumefaciens which are causative agents of crown-gall disease in a wide range of plants. In dual culture assays, the pool of volatiles emitted by the tested Pseudomonas and Serratia strains suppressed the formation of biofilms of A. tumefaciens strains grown on polycarbonate membrane filters and killed Agrobacterium cells in mature biofilms. The individual VOCs produced by the tested Pseudomonas strains, that is, ketones (2-nonanone, 2-heptanone, 2-undecanone), and dimethyl disulfide (DMDS) produced by Serratia strains, were shown to kill A. tumefaciens cells in mature biofilms and suppress their formation. The data obtained in this study suggest an additional potential of some ketones and DMDS as protectors of plants against A. tumefaciens strains, whose virulence is associated with the formation of biofilms on the infected plants. PMID:27214244

  1. Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens

    SciTech Connect

    Muller, A.; Manzara, T.; Lurquin, P.F.

    1984-09-17

    Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype. In vitro crown gall transformation of dicotyledonous plant cells has been demonstrated after cocultivation of cell-wall regenerating mesophyll protoplasts with Agrobacterium tumefaciens cells. In addition, it has been shown that protoplasts freshly isolated from suspension cultures, when treated with A. tumefaciens spheroplasts and a fusogen, also generated cells displaying a typical crown gall phenotype, i.e., phytohormone-independent growth and opine synthesis. Subsequently, both techniques were used to transfer and express foreign genes in plant cells via A. tumefaciens T-DNA integration. For practical purposes, it would be advantageous to be able to perform crown gall transformation of plant cells in tissue culture. The authors report here for the first time the production of Nicotiana tabacum crown gall cells after cocultivation of callus tissue with A. tumefaciens A136 cells. 11 references, 1 figure, 1 table.

  2. Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens.

    PubMed

    Kado, Clarence I

    2014-01-01

    The plant tumor disease known as crown gall was not called by that name until more recent times. Galls on plants were described by Malpighi (1679) who believed that these extraordinary growth are spontaneously produced. Agrobacterium was first isolated from tumors in 1897 by Fridiano Cavara in Napoli, Italy. After this bacterium was recognized to be the cause of crown gall disease, questions were raised on the mechanism by which it caused tumors on a variety of plants. Numerous very detailed studies led to the identification of Agrobacterium tumefaciens as the causal bacterium that cleverly transferred a genetic principle to plant host cells and integrated it into their chromosomes. Such studies have led to a variety of sophisticated mechanisms used by this organism to aid in its survival against competing microorganisms. Knowledge gained from these fundamental discoveries has opened many avenues for researchers to examine their primary organisms of study for similar mechanisms of pathogenesis in both plants and animals. These discoveries also advanced the genetic engineering of domesticated plants for improved food and fiber. PMID:25147542

  3. Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens

    PubMed Central

    Kado, Clarence I.

    2014-01-01

    The plant tumor disease known as crown gall was not called by that name until more recent times. Galls on plants were described by Malpighi (1679) who believed that these extraordinary growth are spontaneously produced. Agrobacterium was first isolated from tumors in 1897 by Fridiano Cavara in Napoli, Italy. After this bacterium was recognized to be the cause of crown gall disease, questions were raised on the mechanism by which it caused tumors on a variety of plants. Numerous very detailed studies led to the identification of Agrobacterium tumefaciens as the causal bacterium that cleverly transferred a genetic principle to plant host cells and integrated it into their chromosomes. Such studies have led to a variety of sophisticated mechanisms used by this organism to aid in its survival against competing microorganisms. Knowledge gained from these fundamental discoveries has opened many avenues for researchers to examine their primary organisms of study for similar mechanisms of pathogenesis in both plants and animals. These discoveries also advanced the genetic engineering of domesticated plants for improved food and fiber. PMID:25147542

  4. Agrobacterium infection and plant defense—transformation success hangs by a thread

    PubMed Central

    Pitzschke, Andrea

    2013-01-01

    The value of Agrobacterium tumefaciens for plant molecular biologists cannot be appreciated enough. This soil-borne pathogen has the unique capability to transfer DNA (T-DNA) into plant systems. Gene transfer involves both bacterial and host factors, and it is the orchestration of these factors that determines the success of transformation. Some plant species readily accept integration of foreign DNA, while others are recalcitrant. The timing and intensity of the microbially activated host defense repertoire sets the switch to “yes” or “no.” This repertoire is comprised of the specific induction of mitogen-activated protein kinases (MAPKs), defense gene expression, production of reactive oxygen species (ROS) and hormonal adjustments. Agrobacterium tumefaciens abuses components of the host immunity system it mimics plant protein functions and manipulates hormone levels to bypass or override plant defenses. A better understanding of the ongoing molecular battle between agrobacteria and attacked hosts paves the way toward developing transformation protocols for recalcitrant plant species. This review highlights recent findings in agrobacterial transformation research conducted in diverse plant species. Efficiency-limiting factors, both of plant and bacterial origin, are summarized and discussed in a thought-provoking manner. PMID:24391655

  5. The Agrobacterium VirE3 effector protein: a potential plant transcriptional activator.

    PubMed

    García-Rodríguez, Fernando M; Schrammeijer, Barbara; Hooykaas, Paul J J

    2006-01-01

    During the infection of plants, Agrobacterium tumefaciens introduces several Virulence proteins including VirE2, VirF, VirD5 and VirE3 into plant cells in addition to the T-DNA. Here, we report that double mutation of virF and virE3 leads to strongly diminished tumor formation on tobacco, tomato and sunflower. The VirE3 protein is translated from a polycistronic mRNA containing the virE1, virE2 and virE3 genes, in Agrobacterium. The VirE3 protein has nuclear localization sequences, which suggests that it is transported into the plant cell nucleus upon translocation. Indeed we show here that VirE3 interacts in vitro with importin-alpha and that a VirE3-GFP fusion protein is localized in the nucleus. VirE3 also interacts with two other proteins, viz. pCsn5, a component of the COP9 signalosome and pBrp, a plant specific general transcription factor belonging to the TFIIB family. We found that VirE3 is able to induce transcription in yeast when bound to DNA through the GAL4-BD. Our data indicate that the translocated effector protein VirE3 is transported into the nucleus and there it may interact with the transcription factor pBrp to induce the expression of genes needed for tumor development. PMID:17130174

  6. Agrobacterium VirD2 protein interacts with plant host cyclophilins.

    PubMed

    Deng, W; Chen, L; Wood, D W; Metcalfe, T; Liang, X; Gordon, M P; Comai, L; Nester, E W

    1998-06-01

    Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer. PMID:9618535

  7. An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells.

    PubMed

    Dumas, F; Duckely, M; Pelczar, P; Van Gelder, P; Hohn, B

    2001-01-16

    Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer. T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2. The VirE2 protein is highly induced on contact of A. tumefaciens with a plant host and has been reported to act in late steps of transfer. One of its previously demonstrated functions is binding to the single-stranded (ss) T-DNA and protecting it from degradation. Recent experiments suggest other functions of the protein. A combination of planar lipid bilayer experiments, vesicle swelling assays, and DNA transport experiments demonstrated that VirE2 can insert itself into artificial membranes and form channels. These channels are voltage gated, anion selective, and single-stranded DNA-specific and can facilitate the efficient transport of single-stranded DNA through membranes. These experiments demonstrate a VirE2 function as a transmembrane DNA transporter, which could have applications in gene delivery systems. PMID:11149937

  8. Agrobacterium-mediated genetic transformation using cotyledons in Japanese pear (Pyrus pyrifolia)

    PubMed Central

    Nakajima, Ikuko; Sato, Yoshihiko; Saito, Toshihiro; Moriguchi, Takaya; Yamamoto, Toshiya

    2013-01-01

    Genetic transformation was successfully established producing both transformed adventitious shoots and calli in Japanese pear (Pyrus pyrifolia Nakai) by using cotyledons as explants. Cotyledons of five cultivars were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying the pBIN19-sgfp, which contained a green fluorescent protein gene and the neomycin phosphotransferase gene. In order to increase transformation efficiency, sonication and ethylenedioxybis (ethylamine)-N,N,N′,N′-tetraacetic acid (EGTA) treatments were applied, which could produce physical wounds across the tissue and prevent plant defense reaction, respectively. Green fluorescent protein (GFP) fluorescence was evaluated two weeks and five months after Agrobacterium inoculation as measures of transient and stable transformations, respectively. As a result, sonication significantly increased both transient and stable expression of GFP fluorescence, whereas EGTA treatment did not show a positive effect on either. Out of 18 regenerated plantlets obtained, one plant regenerated from ‘Agenosho Shinanashi’ showed stable GFP fluorescence. This plant was confirmed as a transformant by PCR and genomic Southern blotting. Three other transformed regenerated shoots by myb gene showed red color, which were derived from ‘Imamuraaki’ by the same transformation method. Transformation system in this study was shown to be reproducible since plural transformants were obtained. PMID:24273422

  9. X-ray structure of imidazolonepropionase from Agrobacterium tumefaciens at 1.87 Å resolution

    SciTech Connect

    Tyagi, Rajiv; Kumaran, Desigan; Burley, Stephen K.; Swaminathan, Subramanyam

    2010-01-12

    Histidine degradation in Agrobacterium tumefaciens involves four enzymes, including histidase (EC 4.3.1.3), urocanase (EC 4.2.1.49), imidazolonepropionase (EC 3.5.2.7), and N-formylglutamate amidohydrolase (EC 3.5.3.8). The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-L-glutamate. Initial studies of the role of imidazolonepropionase in histidine degradation were published in 1953. Subsequent publications have been limited to enzyme kinetics, crystallization, and a recently reported structure determination. The imidazolonepropionases are members of metallodepenent-hydrolases (or amidohydroase) superfamily, which includs ureases, adenosine deaminases, phosphotriesterases, dihydroorotases, allantoinases, hydantoinases, adenine and cytosine deaminases, imidazolonepropionases, aryldial-kylphosphatases, chlorohydrolases, and formylmethanofuran dehydroases. Proteins belonging to this large group share a common three-dimensional structural motif (an eightfold {alpha}/{beta} or TIM barrel) with similar active sites. Most superfamily members also share a conserved metal binding site, involving four histidine residues and one aspartic acid. Imidazolonepropionase is one of the targets selected for X-ray crystallpgrahpic structure determination by the New York Structural GenomiX Research Consortium (NYSGXRC) Target ID: 9252b to correlate the structure function relationship of poorly studied by important enzyme. Here they report the crystal structure of imidazolonepropionase from Agrobacterium tumefaciens determined at 1.87 {angstrom} resolution.

  10. A cytometry microparticle platform approach for screening tobacco microRNA changes after agrobacterium delivery.

    PubMed

    Powell, Joshua D; Chen, Qiang; Mason, Hugh S

    2016-08-01

    MicroRNAs are a class of non-coding regulatory RNAs that can modulate development as well as alter innate antiviral defenses in plants. In this study we explored changes in Nicotiana benthamiana tobacco microRNA expression as it relates to expression of a recombinant anti-Ebola GP1 antibody. The antibody was delivered to tobacco leaves through a bacterial Agrobacterium tumefaciens "agroinfiltration" expression strategy. A multiplex microparticle-based cytometry assay tracked the expression changes of 53 host tobacco microRNAs. Our results revealed that the most abundant microRNAs in actively growing leaves corresponded to nanoparticle probes specific to nta-mir-6149 and nta-miR-168b. After agroinfiltration, probes specific for nta-mir-398, and nta-mir-482d were significantly altered in their respective expression levels, however changes were partially attributed to the infiltration broth medium used in the antibody delivery process. Confirmation of nta-mir-398 and nta-mir-482d expression changes was also verified through RT-qPCR. To our knowledge this study is the first to profile medium and Agrobacterium injection at the microRNA level through a multiplex microparticle approach. PMID:27343681

  11. DNA METHYLATION ANALYSIS DURING THE OPTIMIZATION OF Agrobacterium-MEDIATED TRANSFORMATION OF SOYBEAN.

    PubMed

    Jiang, J; Wing, V; Xiet, T; Shi, X; Wang, Y P; Sokolov, V

    2016-01-01

    Soybean is recognized as one of the plants which are very difficult to be transformed. Considering the low transformation efficiency of soybean, we aimed to determine the effect of 6-benzylaminopurine (6-BA), shoot induction time, and infection time of Agrobacterium on the clonal propagation of Glycine max. Results showed that 1.6 mg/L 6-BA could be optimal to promote the induction of adventitious shoots. An induction time of 15 d was considered optimal for the actual experiment involving soybean shoot induction. Agrobacterium was cultured until an OD600 = 0.8 was reached for an infection time of 30 min; this infection time may be optimal to promote soybean transformation. Whole genome DNA methylation was analyzed by high-performance liquid chromatography (HPLC)-assisted quantification, and DNA methylation result is consistent with the phenotypic data of shoot development. In addition, two methylation-related genes (Decrease in DNA methylation 1 and DNA methyltransferases chromomethylase 2) were analyzed to determine expression differences by qRT-PCR in the shoots that were developed under different experimental conditions. In general, the expression values of these genes were normally downregulated under the recommended experimental conditions of soybean regeneration. This study showed the overall methylation changes in the in vitro culture of soybean, as affected by several variable parameters, which is useful to promote the transformation efficiency of soybean. PMID:27183794

  12. Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens.

    PubMed

    Otten, L; De Greve, H; Hernalsteens, J P; Van Montagu, M; Schieder, O; Straub, J; Schell, J

    1981-01-01

    Insertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity, rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity. Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.

  13. vir-Gene-inducing activities of hydroxycinnamic acid amides in Agrobacterium tumefaciens.

    PubMed

    Berthelot, K; Buret, D; Guerin, B; Delay, D; Negrel, J; Delmotte, F M

    1998-11-20

    Expression of Agrobacterium tumefaciens virulence genes and transformation of dicots by this organism are dependent upon host plant phenolic compounds. Several alkylsyringamides have recently been shown to be powerful inducers of these vir-genes. These synthetic amides, and especially ethylsyringamide, are much stronger inducers than syringic acid. In this work, four alkylamides derived from ferulic or sinapic acids were synthesized by a dicyclohexylcarbodiimide method and tested for their potential to induce vir-gene expression on A. tumefaciens strains harbouring virB::lacZ or virE::lacZ fusion plasmids. Their effectiveness was compared to that of ethylsyringamide and tyraminylferulamide, a naturally occurring amide in plants. Whatever the amine moiety of the amide (ethylamine, propylamine, tyramine or beta-alanine ethyl ester) conjugation of the acid functional group clearly diminished the toxicity to the bacteria of the respective acid at high concentration and thereby increased the vir-inducing potential. However, none of the inducers tested exhibited higher activity than acetosyringone, the reference compound for vir-gene induction, with the exception of ethylsyringamide at concentrations above 1mM. When tested on Agrobacterium tumefaciens strain A348(pSM243cd), ethylferulamide and ethylsinapamide were more efficient than the corresponding phenolic acids but only above 100 microM. PMID:11711062

  14. Infiltration with Agrobacterium tumefaciens induces host defense and development-dependent responses in the infiltrated zone.

    PubMed

    Pruss, Gail J; Nester, Eugene W; Vance, Vicki

    2008-12-01

    Despite the widespread use of Agrobacterium tumefaciens to transfer genes into plant systems, host responses to this plant pathogen are not well understood. The present study shows that disarmed strains of Agrobacterium induce distinct host responses when infiltrated into leaves of Nicotiana tabacum. The responses are limited to the infiltrated zone and consist of i) induction of pathogenesis-related (PR) gene PR-1 expression and resistance to subsequent infection with tobacco mosaic virus, ii) chlorosis and loss of chloroplast rRNAs, and iii) inhibition of leaf expansion. Induction of the latter two sets of responses depends on the age of the leaf and is most apparent in young leaves. Strains with or without binary vectors induce all the responses, showing that DNA transfer is neither required nor inhibitory. A. tumefaciens cured of the tumor-inducing (Ti) plasmid is slightly defective for induction of the three responses, showing that Ti plasmid-encoded factors produced by the disarmed strains contribute only slightly. However, T-DNA-encoded factors alter at least one of the host responses, because infiltration with the oncogenic strain C58 induced more pronounced chlorosis than the disarmed control. Auxin is one of the T-DNA products responsible for disease induction by oncogenic A. tumefaciens. We found that C58-infiltrated zones-but not those infiltrated with the disarmed control-have increased levels of miR393, a microRNA that represses auxin signaling and contributes to antibacterial resistance. PMID:18986249

  15. Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods.

    PubMed

    Joyce, Priya; Hermann, Scott; O'Connell, Anthony; Dinh, Quang; Shumbe, Leonard; Lakshmanan, Prakash

    2014-05-01

    Future genetic improvement of sugarcane depends, in part, on the ability to produce high-yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium-mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5', 3' or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques. PMID:24330327

  16. An efficient Agrobacterium-mediated transformation of strawberry cv. Camarosa by a dual plasmid system.

    PubMed

    Haddadi, Fatemeh; Aziz, Maheran Abd; Abdullah, Siti Nor Akmar; Tan, Soon Guan; Kamaladini, Hossein

    2015-02-23

    An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

  17. Agrobacterium tumefaciens-mediated transformation of the lichen fungus, Umbilicaria muehlenbergii.

    PubMed

    Park, Sook-Young; Jeong, Min-Hye; Wang, Hai-Ying; Kim, Jung A; Yu, Nan-Hee; Kim, Sungbeom; Cheong, Yong Hwa; Kang, Seogchan; Lee, Yong-Hwan; Hur, Jae-Seoun

    2013-01-01

    Transformation-mediated mutagenesis in both targeted and random manners has been widely applied to decipher gene function in diverse fungi. However, a transformation system has not yet been established for lichen fungi, severely limiting our ability to study their biology and mechanism underpinning symbiosis via gene manipulation. Here, we report the first successful transformation of the lichen fungus, Umbilicaria muehlenbergii, via the use of Agrobacterium tumefaciens. We generated a total of 918 transformants employing a binary vector that carries the hygromycin B phosphotransferase gene as a selection marker and the enhanced green fluorescent protein gene for labeling transformants. Randomly selected transformants appeared mitotically stable, based on their maintenance of hygromycin B resistance after five generations of growth without selection. Genomic Southern blot showed that 88% of 784 transformants contained a single T-DNA insert in their genome. A number of putative mutants affected in colony color, size, and/or morphology were found among these transformants, supporting the utility of Agrobacterium tumefaciens-mediated transformation (ATMT) for random insertional mutagenesis of U. muehlenbergii. This ATMT approach potentially offers a systematic gene functional study with genome sequences of U. muehlenbergii that is currently underway. PMID:24386304

  18. Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes.

    PubMed

    Guo, M; Zhang, Y L; Meng, Z J; Jiang, J

    2012-03-16

    Micro-Tom is the smallest known variety of tomatoes. An orthogonal experimental design L(16) (4(5)) was used to optimize Agrobacterium-mediated transformation of cotyledon explants of Lycopersicon esculentum cv. Micro-Tom. Four parameters were investigated to determine their effect on transformation frequency: the concentration of bacterial suspension, time of dip in bacterial suspension, co-cultivation time, and concentration of carbenicillin. We also examined the effect of these parameters on contamination rate, necrosis rate, mortality, cut-surface browning rate, and undamaged explant rate. Both the bacterial and carbenicillin concentrations had a significant influence on the rate of infected explants. The time of co-cultivation also had a significant influence on the transformation parameters. The optimal transformation protocol consisted of an Agrobacterium suspension of 0.5 × 10(8) cells/mL (OD(600) = 0.5) and an infection time of 5 min, one day of co-cultivation and 500 mg/L carbenicillin. Under these conditions, the transformation efficiency of the shoots reached 5.1%; the mean transformation frequency was 3.9% (N = 838).

  19. Stable Agrobacterium-mediated transformation of maritime pine based on kanamycin selection.

    PubMed

    Alvarez, José M; Ordás, Ricardo J

    2013-01-01

    An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β -glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL(-1) kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD(600 nm)) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.

  20. DNA METHYLATION ANALYSIS DURING THE OPTIMIZATION OF Agrobacterium-MEDIATED TRANSFORMATION OF SOYBEAN.

    PubMed

    Jiang, J; Wing, V; Xiet, T; Shi, X; Wang, Y P; Sokolov, V

    2016-01-01

    Soybean is recognized as one of the plants which are very difficult to be transformed. Considering the low transformation efficiency of soybean, we aimed to determine the effect of 6-benzylaminopurine (6-BA), shoot induction time, and infection time of Agrobacterium on the clonal propagation of Glycine max. Results showed that 1.6 mg/L 6-BA could be optimal to promote the induction of adventitious shoots. An induction time of 15 d was considered optimal for the actual experiment involving soybean shoot induction. Agrobacterium was cultured until an OD600 = 0.8 was reached for an infection time of 30 min; this infection time may be optimal to promote soybean transformation. Whole genome DNA methylation was analyzed by high-performance liquid chromatography (HPLC)-assisted quantification, and DNA methylation result is consistent with the phenotypic data of shoot development. In addition, two methylation-related genes (Decrease in DNA methylation 1 and DNA methyltransferases chromomethylase 2) were analyzed to determine expression differences by qRT-PCR in the shoots that were developed under different experimental conditions. In general, the expression values of these genes were normally downregulated under the recommended experimental conditions of soybean regeneration. This study showed the overall methylation changes in the in vitro culture of soybean, as affected by several variable parameters, which is useful to promote the transformation efficiency of soybean.