Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically.

In rapidly growing Escherichia coli cells replication cycles overlap and initiation occurs at multiple replication origins (oriCs). All origins within a cell are initiated essentially in synchrony and only once per cell cycle. Immediate re-initiation of new origins is avoided by sequestration, a mechanism dependent on the SeqA protein and Dam methylation of GATC sites in oriC. Here, GATC sites in oriC were changed to GTTC. This reduced the sequestration to essentially the level found in SeqA-less cells. The mutant origins underwent re-initiation, showing that the GATC sites in oriC are required for sequestration. Each re-initiation eventually gave rise to a cell containing an extra nucleoid. The three-nucleoid cells displayed one asymmetrically placed FtsZ-ring and divided into a two-nucleoid cell and a one-nucleoid cell. The three nucleoid-cells thus divided into three daughters by two consecutive divisions. The results show that extra rounds of replication cause extra daughter cells to be formed prematurely. The fairly normal mutant growth rate and size distribution show, however, that premature rounds of replication, chromosome segregation, and cell division are flexibly accommodated by the existing cell cycle controls.