scholarly journals Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

1996 ◽  
Vol 178 (7) ◽  
pp. 2086-2093 ◽  
Author(s):  
P A Sobecky ◽  
C L Easter ◽  
P D Bear ◽  
D R Helinski
Keyword(s):  
Host Range ◽  
Broad Host Range ◽  
Broad Host Range Plasmid ◽  
Stable Maintenance ◽  
Plasmid Rk2

scholarly journals Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

1998 ◽  
Vol 180 (22) ◽  
pp. 6023-6030 ◽  
Author(s):  
Carla L. Easter ◽  
Helmut Schwab ◽  
Donald R. Helinski
Keyword(s):  
Host Range ◽  
Plasmid Stability ◽  
Broad Host Range ◽  
E Coli ◽  
Broad Host Range Plasmid ◽  
Resolution System ◽  
Stable Maintenance ◽  
Plasmid Stabilization ◽  
Plasmid Rk2

ABSTRACT The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons,parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, andparCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or bothparB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss ofparCBA activity. For P. aeruginosaPAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosaPAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parAand res alone were sufficient for stabilization. Thecer resolvase system of plasmid ColE1 and theloxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all threeE. coli strains. These observations indicate that theparA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of theparC and parB genes but that in at least oneE. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or thecer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.

scholarly journals Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

Genetics ◽  
1992 ◽  
Vol 130 (1) ◽  
pp. 27-36 ◽  
Author(s):  
A Greener ◽  
S M Lehman ◽  
D R Helinski
Keyword(s):  
Host Range ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Firefly Luciferase ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Broad Host Range Plasmid ◽  
Transcriptional Start Sites ◽  
Plasmid Rk2

Abstract A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2

1982 ◽  
Vol 152 (3) ◽  
pp. 1078-1090
Author(s):  
R Meyer ◽  
M Hinds
Keyword(s):  
Dna Replication ◽  
Host Range ◽  
Plasmid Dna ◽  
Plasmid Vector ◽  
Broad Host Range ◽  
Plasmid Maintenance ◽  
Broad Host Range Plasmid ◽  
Expression Of Genes ◽  
Plasmid Partitioning ◽  
Plasmid Rk2

By cloning fragments of plasmid DNA, we have shown that RK2 expresses incompatibility by more than one mechanism. One previously identified (R. J. Meyer, Mol. Gen, Genet. 177:155--161, 1979; Thomas et al., Mol. Gen. Genet. 181:1--7, 1981) determinant for incompatibility is linked to the origin of plasmid DNA replication. When cloned into a plasmid vector, this determinant prevents the stable inheritance of a coresident RK2. However, susceptibility to this mechanism of incompatibility requires an active RK2 replicon and is abolished if another replicator is provided. We have also cloned a second incompatibility determinant, encoded within the 54.1- to 56.4-kilobase region of RK2 DNA, which we call IncP-1(II). An RK2 derivative remains sensitive to IncP-1(II), even when it is not replicating by means of the RK2 replicon. The 54.1- to 56.4-kilobase DNA does not confer susceptibility to the IncP-1(II) mechanism, nor does it encode a detectable system for efficient plasmid partitioning. The incompatibility may be related to the expression of genes mapping in the 54.1- to 56.4-kilobase region, which are required for plasmid maintenance and suppression of plasmid-encoded killing functions.

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