scholarly journals IncC of Broad-Host-Range Plasmid RK2 Modulates KorB Transcriptional Repressor Activity In Vivo and Operator Binding In Vitro

1999 ◽  
Vol 181 (9) ◽  
pp. 2807-2815 ◽  
Author(s):  
Grazyna Jagura-Burdzy ◽  
Kalliope Kostelidou ◽  
Jessica Pole ◽  
Dheeraj Khare ◽  
Anthony Jones ◽  
...  
Keyword(s):  
Host Range ◽  
Broad Host Range ◽  
Coordinate Regulation ◽  
Broad Host Range Plasmid ◽  
Reporter Systems ◽  
Operator Binding ◽  
Repressor Activity ◽  
Plasmid Rk2

ABSTRACT The korAB operon of broad-host-range plasmid RK2 encodes five genes, two of which, incC andkorB, belong to the parA and parBfamilies, respectively, of genome partitioning functions. BothkorB and a third gene, korA, are responsible for coordinate regulation of operons encoding replication, transfer, and stable inheritance functions. Overexpression of incCalone caused rapid displacement of RK2. Using two different reporter systems, we show that incC modulates the action of KorB. Using promoter fusions to the reporter gene xylE, we show that incC potentiates the repression of transcription bykorB. This modulation of korB activity was only observed with incC1, which encodes the full-length IncC (364 amino acids [aa]), whereas no effect was observed withincC2, which encodes a polypeptide of 259 aa that lacks the N-terminal 105 aa. Using bacterial extracts with IncC1 and IncC2 or IncC1 purified through the use of a His6 tail and Ni-agarose chromatography, we showed that IncC1 potentiates the binding of KorB to DNA at representative KorB operators. The ability of IncC to stabilize KorB-DNA complexes suggests that these two proteins work together in the global regulation of many operons on the IncP-1 genomes, as well in plasmid partitioning.

Generation in vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease

Gene ◽  
1976 ◽  
Vol 1 (1) ◽  
pp. 107-119 ◽  
Author(s):  
David Figurski ◽  
Richard Meyer ◽  
Daniel S. Miller ◽  
Donald R. Helinski
Keyword(s):  
Host Range ◽  
Restriction Endonuclease ◽  
Broad Host Range ◽  
Phage Mu ◽  
Broad Host Range Plasmid ◽  
Plasmid Rk2

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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