Sister chromosome cohesion of Escherichia coli.

We analysed Escherichia coli cells synchronized for initiation of chromosomal DNA replication by fluorescence in situ hybridization (FISH) using fluorescent DNA probes corresponding to various chromosomal regions. Sister copies of regions in an approximately oriC-proximal half of the chromosome are cohesive with each other after replication until the late period of chromosome replication. Sister copies of regions relatively close to the terminus are also separated from each other in the same late period of replication. It is important that sister copies in all the tested regions are thus separated from each other nearly all at once in the late period of chromosome replication. These results are consistent with results obtained by FISH in randomly growing cultures. Cohesion of sister copies in an oriC-close region is observed in a dam null mutant lacking DNA adenine methyltransferase the same as in the parental isogenic dam+ strain, indicating that the cohesion is independent of DNA adenine methyltransferase. This further implies that hemimethylated DNA-binding proteins, such as SeqA, are not involved in the cohesion. On the other hand, the cohesion of sister copies of the oriC-close region was not observed in mukB null mutant cells, suggesting that MukB might be involved in the chromosome cohesion.

Complex formation of MukB, MukE and MukF proteins involved in chromosome partitioning in Escherichia coli.

mukF, mukE and mukB genes are essential for the process of chromosome partitioning in Escherichia coli. We have studied protein-protein interactions among MukB, MukE and MukF proteins by co-immunoprecipitation and sucrose gradient sedimentation experiments, using mukFEB null cells harboring plasmids carrying the wild-type or mutant-type mukFEB operon. MukB forms a complex with MukF and MukE. Analysis of mutant MukB proteins suggested that MukF and MukE bind the C-terminal globular domain of MukB. MukF is indispensable for an interaction between MukB and MukE; however, MukF itself is able to associate with MukB even in the absence of MukE. We have also found that MukF has a Ca(2+)-binding activity. Although purified MukF was able to make a complex either with MukE or MukB, a complex consisting of the three Muk proteins was barely detected in vitro. However, increasing the Ca(2+) or Mg(2+) concentration in the reaction partially restored complex formation. This suggests that Ca(2+) or Mg(2+) may be required for the formation of a complex consisting of the three Muk proteins, and thus may participate in a particular step during chromosome partitioning.