Conditional lethal mutants of the kilB determinant of broad host range plasmid RK2.
We have examined the relationship of kilB to the other known determinants which map in the 14'-22' region of RK2. These are trfA, which encodes a diffusible replication function, and tra3, which specifies a function required for plasmid transmissibility. We found that, in addition to kilB, both tra3 and trfA functions are expressed by the cloned 14'-22' region of RK2. Four temperature-sensitive mutants of kilB were isolated by in vitro mutagenesis of the cloned segment. At 42 degrees C these mutant plasmids can be maintained in Escherichia coli cells which lack a korB+ helper plasmid. At 30 degrees C the helper plasmid is required. Our analysis of these mutants revealed that kilB function is distinct from those of trfA and tra3. One mutant plasmid was temperature-sensitive for maintenance of an RK2 ori plasmid, but this phenotype was shown to be independent of the KilB(ts) phenotype. Thus, kilB appears to be a separate new locus in this portion of the RK2 genome. In addition, these mutants allowed us to test for the existence of an essential replication determinant (trfB) in the 50.4'-56.4' region of RK2. Our results demonstrate that this region is non-essential for replication from the RK2 ori in E. coli. We propose an alternative hypothesis to explain the role of the RK2 trfB region for plasmid maintenance in E. coli.
D002874:Chromosome Mapping; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005804:Genes, Lethal; D005816:Genetic Complementation Test; D009154:Mutation; D010957:Plasmids; D013696:Temperature; D014170:Transformation, Genetic
D004269:DNA, Bacterial