RESUMO
pPS10 is a replicon isolated from Pseudomonas syringe pv. savastanoi that can be established at 37 degrees C efficiently in Pseudomonas aeruginosa but very inefficiently in Escherichia coli. The establishment of the wild-type pPS10 replicon in E. coli is favored at low temperatures (30 degrees C or below). RepA protein of pPS10 promotes in vitro plasmid replication in extracts from E. coli, and this replication depends on host proteins DnaA, DnaB, DnaG, and SSB. Mutant plasmids able to efficiently replicate in E. coli at 37 degrees C were obtained. Three of four mutants whose mutations were mapped show a conservative Ala-->Val change in the amino-terminal region of the replication protein RepA. Plasmids carrying this mutation maintain the capacity to replicate in P. aeruginosa and have a fourfold increase in copy number in this host. The mutation does not substantially alter the autoregulation mediated by RepA. These results show that the physiological conditions of the host as well as subtle changes in the plasmid replication protein can modulate the host range of the pPS10 replicon.
Assuntos
Proteínas de Bactérias/genética , Sequência Conservada , Escherichia coli/genética , Plasmídeos/genética , Mutação Puntual , Fatores de Transcrição/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Sequência de Bases , Mapeamento Cromossômico , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/fisiologia , Bactérias Gram-Negativas/genética , Homeostase , Dados de Sequência Molecular , Plasmídeos/fisiologia , Pseudomonas aeruginosa/genética , Temperatura , Fatores de Transcrição/biossínteseRESUMO
We have found that DnaA dependent replication of R1 still occurred when 5 of the 9 bases in the dnaA box present in oriR were changed by site directed mutagenesis although the replication efficiency decreased to 20% and 70% of the wild-type origin in vitro and in vivo respectively. Additional mutation of a second dnaA box, 28 bp upstream oriR, that differs in only one base from the consensus sequence, did not affect the level of replication whereas polyclonal antibodies against DnaA totally abolished in vitro replication in the absence of the dnaA box. Wild-type RepA as well as a RepA mutant, RepA2623, that binds to oriR but that is inactive in promoting in vitro replication of plasmid R1, induce efficient binding of DnaA to the dnaA box. However, specific binding of DnaA to oriR was not detected by DNase I protection experiments in the absence of the dnaA box. These results suggest that the entrance of the DnaA protein in oriR is promoted initially by interactions with a RepA-oriR pre-initiation complex and that, in the absence of the dnaA box, these interactions can support, with reduced efficiency, DnaA dependent replication of plasmid R1.
