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TrfA is the only plasmid-encoded protein required for initiation of replication of the broad-host-range plasmid RK2. Here we describe the isolation of fourtrfA mutants temperature sensitive for replication inPseudomonas aeruginosa . One of the mutations led to substitution of arginine 247 with cysteine. This mutant has been previously described to be temperature sensitive for replication, but poorly functional, inEscherichia coli . The remaining three mutants were identical, and each of them carried two mutations, one leading to substitution of arginine 163 with cysteine (mutation 163C) and the other a codon-neutral mutation changing the codon for glycine 235 from GGC to GGU (mutation 235). Neither of the two mutations caused a temperature-sensitive phenotype alone inP. aeruginosa , and the effect of the neutral mutation was caused by its ability to strongly reduce thetrfA expression level. The double mutant and mutant 163C could not be stably maintained inE. coli , but mutant 235 could be established and, surprisingly, displayed a temperature-sensitive phenotype in this host. Mutation 235 strongly reduced thetrfA expression level also inE. coli . The glycine 85 codon intrfA mRNA is GGU, and a change of this to GGC did not significantly affect expression. In addition, we found that wild-typetrfA was expressed at much lower levels inE. coli than inP. aeruginosa , indicating that this level is a key parameter in the determination of the temperature-sensitive phenotypes in different species. TheE. coli lacZ gene was translationally fused at the 3′ end and internally intrfA , in both cases leading to elimination of the effect of mutation 235 on expression. We therefore propose that this mutation acts through an effect on mRNA structure or stability.

journal of bacteriology

Species-Dependent Phenotypes of Replication-Temperature-Sensitive <i>trfA</i> Mutants of Plasmid RK2: a Codon-Neutral Base Substitution Stimulates Temperature Sensitivity by Leading to Reduced Levels of <i>trfA</i> Expression

Species-Dependent Phenotypes of Replication-Temperature-Sensitive trfA Mutants of Plasmid RK2: a Codon-Neutral Base Substitution Stimulates Temperature Sensitivity by Leading to Reduced Levels of trfA Expression