No abstract

P1 ParA is a member of the Walker-type family of partition ATPases involved in the segregation of plasmids and bacterial chromosomes. ATPases of this class interact with DNA non-specifically in vitro and colocalize with the bacterial nucleoid to generate a variety of reported patterns in vivo. Here, we directly visualize ParA binding to DNA using total internal reflection fluorescence microscopy. This activity depends on, and is highly specific for ATP. DNA-binding activity is not coupled to ATP hydrolysis. Rather, ParA undergoes a slow multi-step conformational transition upon ATP binding, which licenses ParA to bind non-specific DNA. The kinetics provide a time-delay switch to allow slow cycling between the DNA binding and non-binding forms of ParA. We propose that this time delay, combined with stimulation of ParA's ATPase activity by ParB bound to the plasmid DNA, generates an uneven distribution of the nucleoid-associated ParA, and provides the motive force for plasmid segregation prior to cell division.

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown. We reconstituted and visualized ParA-mediated plasmid partition inside a DNA-carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB-stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion-ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid. The EMBO Journal (2013Journal ( ) 32, 1238Journal ( -1249Journal ( . doi:10.1038Journal ( / emboj.2013 IntroductionHow is energy used to transport and spatially organize large objects, such as DNA, in a cell? In eukaryotes, a mechanically driven mitotic spindle apparatus separates chromosomes. In bacteria, the most common DNA segregation mechanism employed by chromosomes and plasmids is still unclear. All bacterial chromosomes and most naturally occurring plasmids are of low-copy number, and many employ active segregation (or partition) systems to ensure inheritance. Partition systems in bacteria are minimalistic, involving only three principal components: an NTPase that drives partition, a partition site on the DNA target, and a partition site binding protein that forms a large partition complex on the DNA target. The most prevalent class of partition systems in the microbial world use ParA-type ATPases that carry a deviant Walker-type active site, but the mechanism remains elusive.The plasmids P1 and F are stably maintained in Escherichia coli and their partition systems are paradigms for studying ParA-mediated DNA segregation. The three essential plasmidencoded components are ParA (or F SopA)-the ATPase, ParB (or F SopB)-the partition site binding protein, and parS (or F sopC)-the partition site on the plasmid. ParBs load onto and around their cognate partition site to form partition complexes, which have been observed as punctate foci in vivo by fluorescence microscopy (Hirano et al, 1998;Erdmann et al, 1999;Lim et al, 2005;Adachi et al, 2006;Sengupta et al, 2010). ParA ATPase activity is critical to the partition process (Ebersbach and Gerdes, 2001;Fung et al, 2001;Barilla et al, 2005;Pratto et al, 2008). ParA alone has weak ATPase activity that is mildly stimulated by non-specific DNA (nsDNA) or ParB (Davis et al, 1992;Watanabe et al, 1992). Together, ParB and DNA synergistically stimulate ParA ATPase activity. But...