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.

Modulation of pPS10 host range by DnaA.

Narrow-host-range plasmid pPS10, originally found in Pseudomonas savastanoi, is unable to replicate in other strains such as Escherichia coli. Here, we report that the establishment of pPS10 in E. coli can be achieved by a triple mutation in the dnaA gene of E. coli (dnaA403), leading to Q14amber, P297S and A412V changes in the DnaA host replication protein (DnaA403 mutant). As the E. coli strain used contained double amber suppressor mutations (supE, supF), the amber codon in dnaA403 can be translated into glutamine or tyrosine. Genetic analysis of DnaA proteins containing either the individual changes or their different combinations suggests that the P297S mutation is crucial for the establishment of the pPS10 replicon in E. coli. The data also indicate that the P297S change is toxic to the cell and that the additional mutations in DnaA403 could contribute to neutralize this toxicity. To our knowledge, this work reports the first chromosome mutant described in the literature that allows the host range broadening of a plasmid, highlights the essential role played by DnaA in the establishment of pPS10 replicon in E. coli and provides support for the hypothesis that interactions between RepA and DnaA modulate the establishment of pPS10 in that bacteria and probably in other species.