Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens

Ward, J E; Dale, E M; Nester, E W; Binns, Andrew N.

doi:10.1128/jb.172.9.5200-5210.1990

Cited by 78 publications

(96 citation statements)

References 45 publications

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“…An indication of the variability in fractionation is the different amount of protein recovered in the inner and outer membrane fractions. While Ward et al (55) recovered 33% of the membrane protein in the outer membrane fraction, we recovered 45% in the same fraction. The amino acid sequence of VirB10 has no export signal sequence and at least one membrane-spanning alpha-helix, suggesting an inner membrane localization.…”

Section: Discussionmentioning

confidence: 65%

“…Furthermore, previous studies have shown that at least three VirB proteins were localized to the membranes ofA. tumefaciens (9,16,55). In the present study, we extended those analyses to determine the subcellular location of seven VirB polypeptides, using two different cell fractionation procedures.…”

mentioning

confidence: 64%

“…The amino acid sequence of VirB10 has no export signal sequence and at least one membrane-spanning alpha-helix, suggesting an inner membrane localization. Ward et al (55) suggested that VirB10 may form an aggregate with itself or other proteins localized to the inner membrane. Our results support this suggestion and further suggest that the complex (aggregate) may span both membranes.…”

Section: Discussionmentioning

confidence: 99%

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Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure

1993

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Plant cell transformation by Agrobacteriwun Iwefaciens involves the transfer of a single-stranded DNAprotein complex (T-complex) from the bacterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes. Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions. VirB5 was in both the inner membrane and cytoplasm. Six of the VwrB proteins were detected in the membrane fractions only. Three of these, VWrB8, VirB9, and VWrBlO, were present in both inner and outer membrane fractions regardless of the fractionation method used. Three additional VwrB proteins, VirBi, VWrB4, and VWrB1I, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method. These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate. That most of the VwrB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions. Agrobacterium tumefaciens has the unique ability to transfer DNA to plant cells. In nature, the transfer and expression of a specific fragment of DNA (T-DNA) cause a neoplastic growth on the plant host. In the laboratory, this DNA transfer phenomenon has been exploited by using disarmed vectors to introduce new genes into plants. The T-DNA and the components necessary for packaging and transfer of the single-stranded T-DNA (T-strand) (45) are encoded by the tumor-inducing (Ti) plasmid in A. tumefaciens. The virulence (vir) region contains four operons (virA, virG, virD, and virB) that encode proteins required for virulence on all hosts (44). Three additional operons (virE, virC, and virH) encode proteins that are required only on some hosts (25,44).The transfer of DNA from the bacterium to the plant cell involves at least seven recognizable steps. Of the bacterial components necessary for DNA transfer, only the first step is thought to be mediated by chromosomally encoded functions (reviewed in reference 5). The remaining steps involve proteins that are encoded by the vir region on the Ti plasmid (reviewed in reference 59). The seven steps are as follows: (i) the bacterium binds to a specific component of the plant cell surface; (ii) a bacterial membrane protein (VirA) recognizes signals released by a wounded plant cell and transduces the signal v...

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Section: Discussionmentioning

confidence: 65%

mentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

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Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure

1993

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“…virD4 and the virB operon are not required for any of the early steps such as attachment, induction, or T-DNA processing but are absolutely essential for tumor formation (7,20,51,54,66). VirD4, VirB1, VirB2, VirB3, VirB4, VirB5, VirB8, VirB9, VirB10, and VirB11 have all been localized to the membrane fraction by immunological techniques (12,41,50,51,57,58,67). These proteins could form a pilus-like structure in the membrane that is similar to those involved in conjugation of plasmids between gram-negative bacteria (26,72).…”

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confidence: 99%

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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“…Cell fractionation has shown that eight VirB proteins are membrane proteins [22][23][24] , which is consistent with a role as a channel. The virB1, virB2, virB5, virB6, virB7 and virB10 genes have all been shown to encode exported products [24][25][26] (Table 1). …”

Section: T-dna Transfermentioning

confidence: 99%

Adaptation of a conjugal transfer system for the export of pathogenic macromolecules

Winans

Burns

Christie

1996

Trends in Microbiology

182

146

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Conjugal transfer of bacterial plasmids requires a pore through which DNA can traverse the envelopes of the donor and recipient cells. Recent studies indicate that these pores, which are composed of approximately ten proteins, are evolutionarily related to the transport systems required for the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to plant cells and for toxin secretion from Bordetella pertussis.Nature provides countless examples of the appropriation and adaptation of familiar proteins or groups of proteins to satisfy newly arising needs. One example is the recently discovered adaptation of a conjugal transfer system by two unrelated pathogenic bacteria. Bacterial conjugation, the horizontal transfer of plasmid DNA by cell-to-cell contact, is found in many or perhaps most prokaryotes 1 . It has recently become apparent that the plant pathogen Agrobacterium tumefaciens and the human pathogen Bordetella pertussis have independently coopted a similar set of tra (conjugal transfer system) genes for the export of macromolecules. Agrobacterium tumefaciens uses a conjugal transfer-like system to transfer oncogenic T-DNA to plants, whereas B. pertussis uses a related set of proteins to export one of its important virulence factors, pertussis toxin. Thus, similar systems are used by bacteria to transport both DNA and proteins across bacterial membranes. Self-transmissible plasmidsThe conjugal transfer systems of several self-transmissible plasmids that colonize Gramnegative bacteria have been studied. Plasmids of the N incompatibility (IncN) group direct the synthesis of conjugal pili. Unlike the thick flexible pili of the F plasmid, IncN pili are thin (approximately 10 nm) and brittle, and have pointed tips and basal knobs 2,3 . They are readily detached from bacterial cells and are found predominantly in the culture *

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Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens

Cited by 78 publications

References 45 publications

Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure

Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure

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

Adaptation of a conjugal transfer system for the export of pathogenic macromolecules

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