This is a reply to an article published in a previous issue of Microbiology (4). We first demonstrated cohesion of replicated oriC copies in E. coli cells (temperature sensitive dnaA mutant cells) synchronized for initiation of chromosomal DNA replication [3; this manuscript is not cited in the associated comment article by Nanninga et al.(4)]. In the same manuscript, we also demonstrated that a single oriC focus, which was detected by FISH (fluorescence in situ hybridization), was located at the middle of the cell (more correctly, at the middle of the nucleoid) just before and after initiation of replication. A single oriC focus appeared to separate into two foci late in the chromosome replication cycle. The result suggests that oriC is replicated at the middle of the nucleoid and these sister copies remain closely associated for a long time. Replicated sister copies of other chromosomal regions also remain associated after replication until late in replication (11).

Sister oriC copies tend to locate as a single focus near the middle of the nucleoid until the point that two distinct foci can be seen (3). However, sometimes it appears that a single focus can move to a border of the nucleoid, separate into two foci and these can migrate asymmetrically toward both pole-proximal borders of the nucleoid prior to the cell division as described previously (6). Based on the cohesion model,‘duplication of an oriC focus’(6) does not mean ‘replication of the oriC segment’, but ‘separation of cohesive oriC copies’. Thus the distinction is based on our experimental design in which replication was initiated at a known time by a temperature downshift in the dnaAts strain, and then observation of separate foci was seen only at a much later time. The second subject is translocation of replication apparatuses during chromosome replication. Paired replication apparatuses that are acting for bidirectional replication of the chromosomal DNA are first closely located with each other at the middle of the nucleoid. However these apparatuses separate and rapidly migrate to 1\4 and 3\4 cell positions at the time that replication forks are estimated to have progressed bidirectionally approximately one third of the distance between oriC and terminus. This model, named the ‘translocating replication apparatuses model’(3; Fig. 1) or ‘replication apparatuses translocation model’, is based on analysis of the subcellular localization of SeqA-bound hemimethylated nascent DNA clusters (2, 3, 9, 11; for a review, see 1) and beta-clamp clusters (DNA sliding clump or DnaN) of DNA polymerase III holoenzyme (10). According to this model, copies of oriC and oriC-proximal genes that are replicated before the translocation of replication apparatuses might remain localized as a single focus in the middle of the nucleoid (Fig. 1B-b and c). On the other hand, other genes that are replicated after the translocation of replication apparatuses might be localized as a single focus around the replication apparatus localized at the 1\4 or 3\4 cell position until release from cohesion (Fig. 1B-e and f). Our previous results by FISH (7) are consistent with the above speculation. After release from cohesion, sister chromosomes are rearranged to form two separated nucleoids, in which oriC copies are localized near pole-proxi-