IBBTEC, Instituto de Biomedicina y Biotecnologia de Cantabria (CSIC-UC-SODERCAN), Facultad de Medicina, Universidad de Cantabria, Santander, Spain
Abstract
The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation) and a propagation mode (conjugation). The consequences of this novel concept in plasmid physiology will be discussed.Author Summary
The ability of bacteria to evolve and adapt to new environments most often results from the acquisition of new genes by horizontal transfer. Plasmids have a preponderant role in gene exchanges through their ability to transfer DNA by conjugation, a process that transports DNA between bacteria. Besides, plasmids are autonomous DNA molecules that are faithfully transmitted to cell progeny during vegetative cell multiplication. In this study, we report a system composed of two proteins, StbA and StbB, which act to balance plasmid R388 physiology between two modes: a maintenance mode (vertical transmission) and a propagation mode (horizontal transmission). We demonstrate that StbA is essential to ensure faithful assortment of plasmid copies to daughter cells. In turn, StbB is required for plasmid R388 adequate localization for conjugation. This is the first report of a system which reconciles plasmid segregation and conjugation. Furthermore, R388 belongs to the IncW family of conjugative plasmids, which are of particular interest due to their exceptionally broad host range. We show that the StbAB system is conserved among a wide variety of conjugative plasmids, mainly broad host range plasmids. Thus, the Stb system could constitute an interesting therapeutic target to prevent the spread of adaptive genes.
