Escherichia coli SeqA Structures Relocalize Abruptly upon Termination of Origin Sequestration during Multifork DNA Replication

Abstract

The Escherichia coli SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, E. coli cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration. Then, two abrupt relocalization events occurred within 2–6 minutes and resulted in SeqA foci localized at each of the cell’s quarter positions. Imaging of cells containing an additional fluorescent tag in the origin region showed that SeqA colocalizes with the origin region during sequestration. This indicates that the newly replicated DNA of first one chromosome, and then the other, is moved from midcell to the quarter positions. At the same time, origins are released from sequestration. Our results illustrate that newly replicated sister DNA is segregated pairwise to the new locations. This mode of segregation is in principle different from that of slowly growing bacteria where the newly replicated sister DNA is partitioned to separate cell halves and the decatenation of sisters a prerequisite for, and possibly a mechanistic part of, segregation.

A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy

“…The conventional uorescence microscopy images ( Fig. 2, yellow arrows), as reported previously, 5,6,8,39,40 however the foci were irregular in size and shape (compare Fig. In the PALM images ( Fig.…”

“…By quantifying the expression level of SeqA with western blotting using the anti-SeqA antibody Slater and co-workers report that wild type E. coli grown in minimal media has on average a 1000 SeqA molecules per cell (between 847 and 1435 SeqA molecules per cell depending on the strain and western blotting technique), which is about ten times more than was determined for the SeqA-PAmCherry fusion used in this study. 39 We therefore conclude that the likely underestimated SeqA molecule numbers are not due to changes in expression level as a result of tagging the protein with FPs. Additionally Durisic and coworkers measured that the (in vivo) photoactivation efficiency of PAmCherry is only as little as 45% in Xenopus oocytes, 37 which will also contribute to an underestimation of the absolute molecule numbers.…”

“…18,38 In each analyzed cell large clusters that resemble the typical SeqA foci 5,6,8,39 were selected upon visual inspection and a region of interest was selected around the area of the cluster/focus. 18,38 In each analyzed cell large clusters that resemble the typical SeqA foci 5,6,8,39 were selected upon visual inspection and a region of interest was selected around the area of the cluster/focus.…”

“…S6 †). 5,39 It has been shown that both the SeqA-GFP expressed from plasmid in the presence of native SeqA as well as SeqA-YFP expressed from the genome and replacing the native SeqA retain the ability to sequester the origins of replication, indicating that the fusion of SeqA to FPs does not compromise its biological function. S8 †).…”

Separation of newly replicated bacterial chromosomes: the role ofEscherichia coliTopoisomerase IV

“…SeqA is a negative modulator of replication initiation, which binds to newly replicated, hemimethylated GATC-sites 15-17 . SeqA forms multimeric structures which trail the replication forks dynamically, always binding to the newest DNA 18-21 . The SeqA-DNA complexes are large and typically encompass 100 kb of DNA.…”