Broad-Host-Range Plasmid Cloning Vectors for Gram-Negative Bacteria

Schmidhauser, Thomas J.; Ditta, Gary S.; Helinski, Donald R.

doi:10.1016/b978-0-409-90042-2.50021-0

Cited by 26 publications

(21 citation statements)

References 161 publications

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“…However, assessment of the significance of a particular determinant for the overall stability of the parental plasmid requires the construction of null mutations in an otherwise wild-type plasmid. This approach is particularly useful for the study of stability determinants of broad-host-range plasmids like RK2 because the behavior of miniplasmids or heterologous replicons used as test vectors varies considerably from host to host (45,47).…”

Section: Discussionmentioning

confidence: 99%

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2

et al. 1995

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The par region of the broad-host-range, IncP␣ plasmid RK2 has been implicated as a stability determinant by its ability to enhance the maintenance of mini-RK2 plasmids or heterologous replicons in a growing population of host cells. The region consists of two operons: parCBA, which encodes a multimer resolution system, and parDE, which specifies a postsegregational response mechanism that is toxic to plasmidless segregants. To assess the importance of this region to the stable maintenance of the complete RK2 plasmid in different hosts, we used the vector-mediated excision (VEX) deletion system to specifically remove the entire par region or each operon separately from an otherwise intact RK2 plasmid carrying a lacZ marker. The par region was found to be important to stable maintenance of RK2lac (pRK2526) in Escherichia coli and five other gram-negative hosts (Agrobacterium tumefaciens, Azotobacter vinelandii, Acinetobacter calcoaceticus, Caulobacter crescentus, and Pseudomonas aeruginosa). However, the relative importance of the parCBA and parDE operons varied from host to host. Deletion of parDE had no effect on the maintenance of pRK2526 in A. calcoaceticus, but it severely reduced pRK2526 maintenance in A. vinelandii and resulted in significant instability in the other hosts. Deletion of parCBA did not alter pRK2526 stability in E. coli, A. tumefaciens, or A. vinelandii but severely reduced plasmid maintenance in A. calcoaceticus and P. aeruginosa. In the latter two hosts and C. crescentus, the ⌬parCBA mutant caused a notable reduction in growth rate in the absence of selection for the plasmid, indicating that instability resulting from the absence of parCBA may trigger the postsegregational response mediated by parDE. We also examined the effect of the conjugal transfer system on RK2 maintenance in E. coli. Transfer-defective traJ and traG mutants of pRK2526 were stably maintained in rapidly growing broth cultures. On solid medium, which should be optimal for IncP-mediated conjugation, colonies from cells containing the pRK2526 tra mutants displayed significant numbers of white (Lac ؊ ) sectors on X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) plates, whereas sectors appeared rarely in colonies from tra ؉ plasmid-containing cells. Both the traJ and traG mutations further reduced the maintenance of the already unstable ⌬par derivative. Thus, these experiments with defined mutations in an intact RK2 plasmid have revealed (i) that the par region allows RK2 to adapt to the different requirements for stable maintenance in various hosts and (ii) that conjugal transfer can contribute to the maintenance of RK2 in a growing population, particularly under conditions that are favorable to RK2 transfer.The extraordinary success of plasmids in nature relies largely on plasmid-specified functions that promote their dissemination and ensure their persistence in growing populations of bacteria. The highly promiscuous, self-transmissible plasmids of incompatibility group P (IncP␣ and IncP␤) are particularly remarka...

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Section: Discussionmentioning

confidence: 99%

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2

et al. 1995

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“…A number of broad-host-range expression vectors using the E. coli lac or tac promoters that can be used to overexpress genes in A. tumefaciens have been described previously (13,37 …”

Section: Characterizationmentioning

confidence: 99%

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes

et al. 1990

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The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the -9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virBII, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHol insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirBll-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A.tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virBIl genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virBII, the virB9 and virB10 genes are required for tumorigenicity.Infection of susceptible plants by the phytopathogenic bacterium Agrobacterium tumefaciens results in crown gall tumors. The unique disease mechanism involves the transfer of a specific DNA molecule (the T-DNA) from a large bacterial tumor-inducing (Ti) plasmid into the plant cell genome (reviewed in references 3 and 58). Expression of T-DNA genes encoding plant growth hormones results in unregulated plant cell growth and subsequent tumor formation. The proteins which mediate most of the steps in T-DNA processing and mobilization are supplied in trans by a separate, nontransferred section of the Ti plasmid termed the virulence (vir) region. In octopine-type Ti plasmids, the vir region consists of a contiguous ==35-kilobase-pair (kbp) segment containing at least seven transcriptional loci. Mutations in these loci either completely abolish virulence (virA, -B, -D, and -G) or cause attenuated virulence (virC, -E, and -F) (26,27,40).Expression of the vir genes is inducible by phenolic compounds, such as acetosyringone, present in wounded plant tissue. A regulatory system consisting of VirA (a transmembrane sensor) and VirG (a transcriptional activator) regulate vir gene expression (24, 25,44). Following vir induction, the VirDl and VirD2 proteins provide topoisomerase (19) and endonuclease (42, 56) functions that initiate T-DNA processing by introducing a site-and strand-specific nick within conserved 25-bp cis-acting sequences, termed borders, that delineate the ends of the T-DNA molec...

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“…Maintenance of this 60-kb plasmid at a copy number of four to seven copies per chromosome in Escherichia coli is dependent on a specific origin of replication, oriV, and the plasmid-encoded trans-acting replication initiation protein TrfA (2)(3)(4)(5). The TrfA protein and the oriVsequence have been shown to be sufficient for the initiation and control of replication of RK2 in almost all Gram-negative bacteria examined to date (6). A major feature of the nucleotide sequence of RK2 oriV is the presence of eight 17-bp direct repeats (iterons) arranged in two clusters, one with three iterons the other with five iterons (7).…”

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confidence: 99%

Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

Blasina

Kittell

Toukdarian

et al. 1996

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

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Broad-Host-Range Plasmid Cloning Vectors for Gram-Negative Bacteria

Cited by 26 publications

References 161 publications

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes

Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

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