Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4

Ziegelin, Günter; Pansegrau, Werner; Strack, Bettina; Balzer, D; Kröger, Manfred; Kruft,; Lanka, Erich

doi:10.3109/10425179109020786

Cited by 86 publications

(79 citation statements)

References 63 publications

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“…The deduced amino acid sequences of 6 of the trb genes (IncP) and 10 of the trw genes (IncW) share significant sequence identity with the VirB proteins (26,74). Not only are the virB genes similar in sequence to the conjugation genes, but they are also functionally similar.…”

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confidence: 99%

“…Recently, the operons required for pilin biogenesis and conjugation of the broad-host-range plasmids of RP4 (IncP group) and R388 (IncW group) have been sequenced (26,74). The deduced amino acid sequences of 6 of the trb genes (IncP) and 10 of the trw genes (IncW) share significant sequence identity with the VirB proteins (26,74).…”

mentioning

confidence: 99%

“…Two of the proteins in each of these systems contain putative nucleotide-binding sites (Walker boxes) (12,26,65,68,74). The two potential nucleotide-binding proteins in the virB operon, VirB4 and VirB11, are both absolutely required for virulence (7,20,51,54).…”

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

1995

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“…First, the sites of single-stranded DNA nicking at the T-DNA borders of A. tumefaciens and the transfer origin of an IncP conjugative plasmid contain nearly identical sequences (56). Second, the VirD2, VirD4, and many of the VirB proteins share significant homologies with proteins involved in bacterial conjugation (24,29,31,35,61). Third, T-DNA transfer and conjugation have functional similarities.…”

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confidence: 99%

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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“…The connecting link between DNA preparation and the mating bridge may be supplied by the two RK2 proteins TraF and TraG, and their analogs in other systems. In RK2, these two proteins appear to be co-transcribed and co-translated (49,52). The TraG protein has an NTP-binding domain and might thereby provide energy for DNA transport, working together with TraF which has sequences suggesting a role as a DNA processivity factor (53,54).…”

Section: Donor Bacterium and Recipient Cell Contact And Mating Bridgementioning

confidence: 99%

Conjugative transfer in the dissemination of beta-lactam and aminoglycoside resistance

Waters¹

1999

Front Biosci

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The dissemination of antibiotic resistance among pathogenic bacteria can be attributed largely to conjugative DNA transfer. The general category of conjugative transfer includes both bacterial plasmid conjugation and the transfer of nonreplicative conjugative transposons. Prototypes for these two systems are the plasmid RK2 and the conjugative transposon Tn916. To address the long-term problem of the increasing prevalence and severity of antibiotic resistance, strategies aimed against conjugative transfer are needed, but their development will require a greater understanding of conjugative resistance gene acquisition. Overviews of the two conjugative transfer systems are presented, to summarize and compare current concepts. Observations regarding transfer of conjugative transposons is consistent with the prevailing model for plasmid conjugation, that is, by the transfer of a single-stranded DNA molecule from the donor to the recipient bacterium, and the generation of the single strand by rolling circle DNA replication. The relevance of vegetative plasmid replication and host range to the spread of multiple drug resistance is discussed, and clinical examples of conjugative transfer of multiple antibiotic resistance illustrate the severity of the current situation. Possible directions, traditional and innovative, are offered to address the conjugative transfer problem in drug resistance, and potentially to break the cycle of antibiotic development followed by the bacterial resistance gene acquisition.

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Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4

Cited by 86 publications

References 63 publications

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

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

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

Conjugative transfer in the dissemination of beta-lactam and aminoglycoside resistance

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