Host-dependent requirement for specific DnaA boxes for plasmid RK2 replication.

Abstract:

:The replication origin of the broad-host-range plasmid RK2, oriV, contains four DnaA boxes, which bind the DnaA protein isolated from Escherichia coli. Using a transformation assay, mutational analysis of these boxes showed a differential requirement for replication in different Gram-negative bacteria. DnaA boxes 3 and 4 were required in E. coli and Pseudomonas putidabut not as strictly in Azotobacter vinelandii and not at all in P. aeruginosa. In vitro replication results using an extract prepared from E. coli demonstrated that the activity of origin derivatives containing mutations in boxes 3 or 4 or a deletion of all four DnaA boxes could be restored by the addition of increasing amounts of purified DnaA protein. High levels of DnaA protein in the presence of the TrfA protein also resulted in the stimulation of open complex formation and DnaB helicase loading on oriV, even in the absence of the four DnaA boxes. These observations at least raise the possibility that an alternative mechanism of initiation of oriV is being used in the absence of the four DnaA boxes and that this mechanism may be similar to that used in P. aeruginosa, which does not require these four DnaA boxes for replication.

journal_name

Mol Microbiol

journal_title

Molecular microbiology

authors

Doran KS,Helinski DR,Konieczny I

doi

10.1046/j.1365-2958.1999.01491.x

subject

Has Abstract

pub_date

1999-08-01 00:00:00

pages

490-8

issue

3

eissn

0950-382X

issn

1365-2958

pii

mole1491

journal_volume

33

pub_type

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