A segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand DNA endonuclease and ligase

Bhattacharjee, Mrinal K.; Meyer, Richard J.

doi:10.1093/nar/19.5.1129

Cited by 47 publications

(56 citation statements)

References 20 publications

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“…For RP4 and RSF1010, the genes encoding these functions flank the nic sites (5,35). Mutational analysis indicates that trans-acting functions encoded in the regions flanking the pTiC58 oriT are required for conjugal transfer (4).…”

Section: Discussionmentioning

confidence: 99%

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders

Cook

Farrand

1992

J Bacteriol

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Ti plasmids ofAgrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally activeAgrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.The gram-negative phytopathogen Agrobacterium tumefaciens induces crown gall tumors on susceptible plant species by a novel gene-transfer process. Tumorigenicity is dependent upon a large plasmid, called Ti, present in the bacterium. During infection, a portion of the Ti plasmid called the T-region is excised and exported from the agrobacterial cell to a plant cell. Once the T-region is integrated into the plant nuclear genome, expression of transfer DNA (T-DNA)-encoded genes leads to the synthesis of phytohormones and unique nutritional compounds collectively named opines. The overproduction of the plant growth factors causes tumor formation. Opines are secreted from the tumors into the rhizosphere, where they become available as a nutritional source to A. tumefaciens strains carrying an appropriate Ti plasmid encoding functions for opine uptake and catabolism (39).In addition to carrying pathogenicity determinants, Ti plasmids also encode conjugal transfer functions which allow for Ti plasmid transmission to Ti plasmid-less agrobacteria (31). Ti plasmid conjugal transfer is normally repressed but is inducible by an opine subset, the conjugal opines (23, 38). Once a strain ofAgrobacterium has acquired a Ti plasmid, it is capable of utilizing opines as a sole nutritional substrate. Thus, the opines synthesized by the biologically engineered plant cells act as signals to s...

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Section: Discussionmentioning

confidence: 99%

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders

Cook

Farrand

1992

J Bacteriol

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“…The MobA protein can cleave the single oriT DNA strand by itself at nic, and can also religate this DNA (Bhattacharjee and Meyer, 1991;Scherzinger et al, 1993). One function of the relaxosome, then, might be to provide the oriT duplex DNA in the plasmid with sufficient single-stranded character to be acted upon by this protein (Zhang and Meyer, 1995).…”

Section: Introductionmentioning

confidence: 99%

The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer

Zhang

Meyer

1997

Molecular Microbiology

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SummaryThe frequency of conjugal mobilization of plasmid R1162 is decreased approximately 50-fold if donor cells lack MobC, one of the plasmid-encoded proteins making up the relaxosome at the origin of transfer (oriT ). The absence of MobC has several different effects on oriT DNA. Site-and strand-specific nicking by MobA protein is severely reduced, accounting for the lower frequency of mobilization. The localized DNA strand separation required for this nicking is less affected, but becomes more sensitive to the level of active DNA gyrase in the cell. In addition, strand separation is not efficiently extended through the region containing the nick site. These effects suggest a model in which MobC acts as a molecular wedge for the relaxosome-induced melting of oriT DNA. The effect of MobC on strand separation may be partially complemented by the helical distortion induced by supercoiling. However, MobC extends the melted region through the nick site, thus providing the single-stranded substrate required for cleavage by MobA.

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“…plasmids (1,5,7,(13)(14)(15)(16)(17)(18)(19)(20). After the nicking reaction, VirD2 remains associated with the 5' end of the conjugative DNA strand (17)(18)(19).…”

mentioning

confidence: 99%

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

Jasper

Koncz

Schell

et al. 1994

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

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Virulence proteins VirDl and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirDl-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conijugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant ceil nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirDl and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirDl protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirDl, fails to nick linear double-stranded DNA templates in vitro.

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A segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand DNA endonuclease and ligase

Cited by 47 publications

References 20 publications

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders

The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders

The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

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