Few biological systems permit rigorous testing of how changes in DNA sequence give rise to adaptive phenotypes. In this study, we sought a simplified experimental system with a detailed understanding of the genotype-to-phenotype relationship that could be altered by environmental perturbations. We focused on plasmid fitness, i.e., the ability of plasmids to be stably maintained in a bacterial population, which is dictated by the plasmid's replication and segregation machinery. Although plasmid replication depends on host proteins, the type II plasmid partitioning (Par) machinery is entirely plasmid encoded and relies solely on three components: parC, a centromere-like DNA sequence, ParR, a DNA-binding protein that interacts with parC, and ParM, which forms actin-like filaments that push two plasmids away from each other at cell division. Interactions between the Par operons of two related plasmids can cause incompatibility and the reduced transmission of one or both plasmids. We have identified segregation-dependent plasmid incompatibility between the highly divergent Par operons of plasmids pB171 and pCP301. Genetic and biochemical studies revealed that the incompatibility is due to the functional promiscuity of the DNA-binding protein ParRpB171, which interacts with both parC DNA sequences to direct plasmid segregation, indicating that the lack of DNA binding specificity is detrimental to plasmid fitness in this environment. This study therefore successfully utilized plasmid segregation to dissect the molecular interactions between genotype, phenotype, and fitness.
- Bacterial Proteins/metabolism
- DNA, Bacterial/metabolism
- DNA-Binding Proteins/metabolism
- Escherichia coli/genetics
- Genomic Instability