[18] Genetic Analysis of Agrobacterium

Ga, Cangelosi; Ea, Best; Martinetti, Gladys; Ew, Nester

doi:10.1016/0076-6879(91)04020-o

Cited by 237 publications

(213 citation statements)

References 20 publications

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“…When the pH of the LB medium was adjusted to pH 5.5, the medium was designated LB 5.5. Induction broth pH 5.5 (IB 5.5) is a medium that mimics the vir-inducing condition of a plant wound (Cangelosi et al, 1991). When required, medium supplemented with 100 mg carbenicillin ml 21 , 20 mg chloramphenicol ml 21 , 60 mg gentamicin ml 21 or 30 mg kanamycin ml 21 was used for A. tumefaciens.…”

Section: Methodsmentioning

confidence: 99%

Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions

et al. 2014

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Agrobacterium tumefaciens membrane-bound ferritin (MbfA) is a member of the erythrin (Er)–vacuolar iron transport family. The MbfA protein has an Er or ferritin-like domain at its N terminus and has been predicted to have five transmembrane segments in its C-terminal region. Analysis of protein localization using PhoA and LacZ reporter proteins supported the view that the N-terminal di-iron site is located in the cytoplasm whilst the C-terminal end faces the periplasm. An A. tumefaciens mbfA mutant strain had 1.5-fold higher total iron content than the WT strain. Furthermore, multi-copy expression of mbfA reduced total iron content two- and threefold in WT and mbfA mutant backgrounds, respectively. These results suggest that MbfA may function as an iron exporter rather than an iron storage protein. The mbfA mutant showed 10-fold increased sensitivity to the iron-activated antibiotic streptonigrin, implying that the mutant had increased accumulation of intracellular free iron. Growth of the mbfA mutant was reduced in the presence of high iron under acidic conditions. The expression of mbfA was induced highly in cells grown in iron-replete medium at pH 5.5, further supporting the view that mbfA is involved in the response to iron under acidic conditions. A. tumefaciens MbfA may play a protective role against increased free iron in the cytoplasm through iron binding and export, thus preventing iron-induced toxicity via the Fenton reaction.

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Section: Methodsmentioning

confidence: 99%

Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions

et al. 2014

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“…The 3.8-kb BglII fragment containing Mac-CYP2E1-mas 3Ј was subcloned into the BamHI site of the binary vector pCGN1578 (21), which was provided by Luca Comai. The resulting plasmid, pSLD3, was introduced into Agrobacterium by electroporation (22,23).…”

Section: Methodsmentioning

confidence: 99%

Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1

Doty

Shang

Wilson

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

246

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“…Table 1 lists the bacterial strains and plasmids used in this study. DNA was introduced into A. tumefaciens by triparental mating or electroporation using a Bio-Rad Laboratories electroporator (Richmond, Calif.) as described previously (9). E. coli was grown in Luria-Bertani (LB) medium and manipulated as described previously (47).…”

Section: Methodsmentioning

confidence: 99%

“…pJW325 containing virB9 and virB10 was digested with EcoRI to remove a 2.1-kb fragment, which was then ligated into the EcoRI site of pKS29 to generate pKS30. pKS30 was introduced into strain A348 by electroporation, and mutants undergoing marker exchange were selected and analyzed as described previously (9). Strain A10011 was verified to contain nptII and its promoter in place of virB11 by analysis of Southern blots (data not shown) (53).…”

Section: Methodsmentioning

confidence: 99%

“…E. coli was grown in Luria-Bertani (LB) medium and manipulated as described previously (47). A. tumefaciens was grown in MG/L as described previously (9), or vir gene expression was induced by using the following modified procedure. Cells were grown to saturation in MG/L broth in the presence of appropriate antibiotics, pelleted in a microcentrifuge for 2 min at room temperature, washed once with sterile H 2 O, pelleted again, inoculated into a 10-fold volume of modified induction broth [50 mM 2-(N-morpholino)ethanesulfonic acid (MES)-KOH (pH 5.5), AB salts (9), 0.02% yeast extract, 2% glucose, 200 M acetosyringone], and incubated with shaking for 16 h at 28ЊC.…”

Section: Methodsmentioning

confidence: 99%

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Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

1995

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virB11, one of the 11 genes of the virB operon, is absolutely required for transport of T-DNA from Agrobacterium tumefaciens into plant cells. Previous studies reported that VirB11 is an ATPase with autophosphorylation activity and localizes to the inner membrane even though the protein does not contain the consensus N-terminal export sequence. In this report, we show that VirB11 localizes to the inner membrane even in the absence of other tumor-inducing (Ti) plasmid-encoded proteins. To facilitate the further characterization of VirB11, we purified this protein from the soluble fraction of an Escherichia coli extract by fusing VirB11 to the maltose-binding protein. The maltose-binding protein-VirB11 fusion was able to complement a virB11 deletion mutant of A. tumefaciens for tumor formation and also localized properly to the inner membrane of A. tumefaciens. The 72-kDa protein, purified from E. coli, exhibited no autophosphorylation, ATPase activity, or ATP-binding activity. To study the importance of the Walker nucleotide-binding site present in VirB11, mutations were generated to replace the conserved lysine residue with either alanine or arginine. Expression of the virB11K175A mutant gene resulted in an avirulent phenotype, and expression of the virB11K175R mutant gene gave rise to an attenuated virulence phenotype. Both mutant proteins were present at levels three to four times higher than that of VirB11 in the wild-type strain. The mutant genes did not exhibit a transdominant phenotype on tumor formation in bacteria that were expressing wild-type virB11. The mutant proteins also localized properly to the inner membrane of A. tumefaciens, but the VirB11K175R protein appeared to be unstable after lysis of the cells.The soil bacterium Agrobacterium tumefaciens causes the plant disease crown gall. The bacteria can infect dicotyledonous plants at a wound site and induce the formation of tumors at the site of infection. Virulent strains of A. tumefaciens contain a large tumor-inducing (Ti) plasmid that carries the factors responsible for formation of the crown gall tumors. The bacteria have the unusual property of being able to transfer a segment of DNA (T-DNA) from the Ti plasmid into the host plant cells. The T-DNA is integrated into the plant genome, and expression of the oncogenes present in the T-DNA results in formation of the tumor (for recent reviews, see references 26, 70, and 73).The genes required for the transfer of T-DNA into the recipient plant cells are divided into eight vir operons on the octopine-type Ti plasmid (54). These genes are expressed only when the bacteria are exposed to wounded plant cells as a result of the action of several plant signal molecules acting in concert with the virA and virG gene products. Once the vir genes have been expressed, the gene products act in a coordinated fashion to synthesize T-DNA strands and direct the transfer of the T-DNA into a recipient plant cell.The T-DNA and associated proteins are transported through the bacterial inner and outer membranes and thr...

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[18] Genetic Analysis of Agrobacterium

Cited by 237 publications

References 20 publications

Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions

Roles of Agrobacterium tumefaciens membrane-bound ferritin (MbfA) in iron transport and resistance to iron under acidic conditions

Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

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