Characterization of transposon Tn5-facilitated donor strains and development of a chromosomal linkage map for Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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Correction: characterization of the virB operon from Agrobacterium tumefaciens Ti plasmid.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)39628-0 ◽

1990 ◽

Vol 265 (8) ◽

pp. 4768

Author(s):

J E Ward ◽

D E Akiyoshi ◽

D Regier ◽

A Datta ◽

M P Gordon ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Ti Plasmid ◽

Virb Operon

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In Vitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium tumefaciens

Journal of Bacteriology ◽

10.1128/jb.01591-08 ◽

2009 ◽

Vol 191 (7) ◽

pp. 2033-2041 ◽

Cited By ~ 18

Author(s):

Meriyem Aktas ◽

Franz Narberhaus

Keyword(s):

Agrobacterium Tumefaciens ◽

In Vitro Assay ◽

Enzyme Properties ◽

Vitro Assay ◽

Plant Infection ◽

In Vitro Characterization ◽

End Products ◽

And Control

ABSTRACT Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

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The Presence and Characterization of a virF Gene on Agrobacterium vitis Ti Plasmids

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.5.429 ◽

1998 ◽

Vol 11 (5) ◽

pp. 429-433 ◽

Cited By ~ 14

Author(s):

B. Schrammeijer ◽

J. Hemelaar ◽

P. J. J. Hooykaas

Keyword(s):

Amino Acids ◽

Agrobacterium Tumefaciens ◽

Molecular Mass ◽

Promoter Region ◽

Consensus Sequence ◽

Tumor Formation ◽

Nicotiana Glauca ◽

Agrobacterium Vitis ◽

Ti Plasmids

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

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