The beta subunit of DNA polymerase III is essential for negative regulation of the initiator protein, DnaA. DnaA inactivation occurs through accelerated hydrolysis of ATP bound to DnaA; the resulting ADP-DnaA fails to initiate replication. The ability of beta subunit to promote DnaA inactivation depends on its assembly as a sliding clamp on DNA and must be accompanied by a partially purified factor, IdaB protein. DnaA inactivation in the presence of IdaB and DNA polymerase III is further stimulated by DNA synthesis, indicating close linkage between initiator inactivation and replication. In vivo, DnaA predominantly takes on the ADP form in a beta subunit-dependent manner. Thus, the initiator is negatively regulated by action of the replicase, a mechanism that may be key to effective control of the replication cycle.

Summary: The minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large‐scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.

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The replication origin and the initiator protein DnaA are the main targets for regulation of chromosome replication in bacteria. The origin bears multiple DnaA binding sites, while DnaA contains ATP/ADP-binding and DNA-binding domains. When enough ATP-DnaA has accumulated in the cell, an active initiation complex can be formed at the origin resulting in strand opening and recruitment of the replicative helicase. In Escherichia coli, oriC activity is directly regulated by DNA methylation and specific oriC-binding proteins. DnaA activity is regulated by proteins that stimulate ATP-DnaA hydrolysis, yielding inactive ADP-DnaA in a replication-coupled negative-feedback manner, and by DnaA-binding DNA elements that control the subcellular localization of DnaA or stimulate the ADP-to-ATP exchange of the DnaA-bound nucleotide. Regulation of dnaA gene expression is also important for initiation. The principle of replication-coupled negative regulation of DnaA found in E. coli is conserved in eukaryotes as well as in bacteria. Regulations by oriC-binding proteins and dnaA gene expression are also conserved in bacteria.

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