Modulation of pPS10 host range by plasmid-encoded RepA initiator protein.
We report here the isolation and analysis of novel repA host range mutants of pPS10, a plasmid originally found in Pseudomonas savastanoi. Upon hydroxylamine treatment, five plasmid mutants were selected for their establishment in Escherichia coli at 37 degrees C, a temperature at which the wild-type form cannot be established. The mutations were located in different functional regions of the plasmid RepA initiation protein, and the mutants differ in their stable maintenance, copy number, and ability to interact with sequences of the basic replicon. Four of them have broadened their host range, and one of them, unable to replicate in Pseudomonas, has therefore changed its host range. Moreover, the mutants also have increased their replication efficiency in strains other than E. coli such as Pseudomonas putida and Alcaligenes faecalis. None of these mutations drastically changed the structure or thermal stability of the wild-type RepA protein, but in all cases an enhanced interaction with host-encoded DnaA protein was detected by gel filtration chromatography. The effects of the mutations on the functionality of RepA protein are discussed in the framework of a three-dimensional model of the protein. We propose possible explanations for the host range effect of the different repA mutants, including the enhancement of limiting interactions of RepA with specific host replication factors such as DnaA.
Maestro
Beatriz
B
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Sanz
Jesús M
JM
Díaz-Orejas
Ramón
R
Fernández-Tresguerres
Elena
E
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D020560:Proteobacteria / Q000235:genetics; D011549:Pseudomonas / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases