Invited Speaker
Marcelo E. Tolmasky
XerCD-mediated recombination at mwr sites is hypersensitive to DNA supercoiling
The K. pneumoniae multiresistance plasmid pJHCMW1 includes mwr, a Xer recombination site that shows unique characteristics. Efficiency of resolution of mwr-containing plasmid dimers is strongly dependent on the osmotic pressure of the growth medium as determined by growth in medium containing 0.5 and 0% added NaCl. Since negative supercoiling of plasmid DNA increases as the added NaCl concentration in the medium decreases from 0.5 to 0%, we tested if efficiency of Xer recombination at mwr exhibits a higher than normal dependency with the topological state of the DNA. In vitro recombination of plasmids containing directly repeated mwr (pSDC203) and cer (pSDC115) sites showed that while pSDC115 recombined at high levels as long as the DNA was not fully relaxed, recombination at mwr in pSDC203 showed a strong dependence of the recombination efficiency with the negative supercoiling density. These results support a model in which modifications at the level of negative supercoiling density caused by changes in the osmotic pressure of the culture medium affects resolution of mwr-containing plasmid dimers, a property that separates mwr from other Xer recombination target sites.
This work has been co-authored by S. Trigueros, T. Tran, N. Sorto, J. Newmark, S. D. Colloms, D. J. Sherratt, and M. E. Tolmasky