Poster: 70 - Print

Dynamics of the multiresistance pJHCMW1 plasmid

Authors

Abstract

Replication and partition of high copy number plasmids has been intensely studied. However many questions remain to be answered like timing and location of replication as well as how the plasmid molecules are distributed within the host cell through its life cycle. To gain more insight on these questions we investigated pJHCMW1, a multiresistance plasmid isolated from a clinical Klebsiella pneumonia strain that has ColE1-type origin of replication.

Plasmid molecules were visualized by inserting arrays of 48 or 96 copies of tetO into pJHCMW1. The plasmid derivatives were used to transform an Escherichia coli strain harboring a gene coding for TetR fused to yellow fluorescent protein in the chromosome. Plasmid molecules were visualized by fluorescence microscopy expressing of the fluorescent TetR derivative. Fluorescent derivatives of replisome components were used to mark the position and timing of replication events. Copy number of pJHCMW1 was determined by qPCR using lysed cells.

The measured copy number of pJHCMW1 was 9 per chromosome origin, or ~18 per cell. Plasmid molecules were mostly located at the poles, but they accumulated at midcell at the time of the cell cycle when sister nucleoids split. Generation of cells with extended nucleoid-free space at the poles showed that the plasmid molecules tended to occupy all the free space. The number of replisome foci increased in cells carrying the plasmid, while the positioning of these extra foci was often polar coinciding with the positioning of plasmids.

Our results support a model where replication of plasmids do not to occur preferentially at any stage of the cell cycle or location of the cells and the plasmid molecules diffuse randomly through the cytoplasm. However, the nucleoid acts like a barrier restricting the plasmid to nucleoid-free zones

Keywords

plasmid, dna replication, partition, qpcr