Modulation of pPS10 host range by plasmid-encoded RepA initiator protein

Abstract

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.

FIG. 1.

Circular map of plasmid pRG9B containing the basic replicon of plasmids pPS10 (upper left) and pBR322 (lower left) and the Km^r gene from pKT231 (right). The most relevant restriction endonuclease sites are also shown.

FIG. 2.

Copy number of the host range mutants in P. aeruginosa. (A) Autoradiograph from total lysates of P. aeruginosa containing wild-type pBM9B and mutated pBM plasmids hybridized to a repA DNA probe. (B) Copy number of pBM mutants relative to that of pBM9B. Values are the averages obtained from five different experiments and were obtained by autoradiograph densitometry corrected to the same amount of chromosomal DNA in all the samples. pSBM135 is not included, as it cannot be established in P. aeruginosa.

FIG. 3.

Two views (differing in 180° rotation) of the modeled three-dimensional structure of pPS10 RepA protein based on the amino acid similarity with the RepE initiation replication protein of F plasmid, showing the mutated positions. The picture shows the N- and C-terminal winged-helix domains in white and gray, respectively, as well as the connecting turn (red) and the LZ motif (blue). DNA is shown only for presentation purposes. Drawing was performed with the RASMOL program (http://www.umass.edu/microbio/rasmol/index2.htm).

FIG. 4.

Binding of MBPRepA proteins to the oriV region. Increasing quantities of protein (0, 0.43, 0.72, 1.44, 2.88, 4.32, 5.76, 7.20, 8.65, 10.09, 11.53, 12.97, 14.41, 21.61, and 28.82 pmol from left to right) were incubated with 24.5 fmol of the labeled origin probe.

FIG. 5.

Binding of MBPRepA proteins to the promoter operator region. Increasing quantities of protein (0, 0.07, 0.14, 0.29, 0.43, 0.57, 0.72, 0.86, 1.01, 1.29, 1.44, 1.80, 2.16, 2.52, and 2.88 pmol from left to right) were incubated with 8.6 fmol of the labeled operator probe.

FIG. 6.

Dot blot analysis of the interaction of DnaA with wild-type and mutant MBPRepA fusions by gel-filtration chromatography. The exclusion volume of the column is 6.0 ml. Extracts were obtained from E. coli LE392(pUC392) and CC118(pMALRepA). Lane 1, extracts containing DnaA plus MBPRepA wild type; lane 2, DnaA plus MBPRepA(G135S); lane 3, DnaA plus MBPRepA(G44S); lane 4, DnaA plus MBPRepA(R93C); lane 5, DnaA plus MBPRepA(T47I); lane 6, DnaA plus MBPRepA(A31V); lane 7, DnaA without MBPRepA.