Abstract
Chromosome replication in Escherichia coli is in part controlled by three non-coding genomic sequences, DARS1, DARS2, and datA that modulate the activity of the initiator protein DnaA. The relative distance from oriC to the non-coding regions are conserved among E. coli species, despite large variations in genome size. Here we use a combination of i) site directed translocation of each region to new positions on the bacterial chromosome and ii) random transposon mediated translocation followed by culture evolution, to show genetic evidence for the importance of position. Here we provide evidence that the genomic locations of these regulatory sequences are important for cell cycle control and bacterial fitness. In addition, our work shows that the functionally redundant DARS1 and DARS2 regions play different roles in replication control. DARS1 is mainly involved in maintaining the origin concentration, whether DARS2 is also involved in maintaining single cell synchrony.
Highlights
The circular chromosome of Escherichia coli is replicated bidirectionally from a single origin, oriC
Replication of the E. coli chromosome is the central event in the cell cycle, with the control of replication enforced at the level of initiation
We show that the chromosomal position of datA, DARS1, and especially DARS2 relative to oriC, is important for cell cycle control and bacterial fitness
Summary
The circular chromosome of Escherichia coli is replicated bidirectionally from a single origin, oriC. The DnaA protein is responsible for replication initiation [1]. DnaA belongs to the AAA+ (ATPases Associated with diverse Activities) proteins and can bind both ATP and ADP with similar high affinities[1]. DnaA is active in replication when bound to ATP (DnaAATP)[2] and facilitates the unwinding of oriC [3,4,5]. After initiation DnaAATP is inactivated, i.e. converted to DnaAADP, by RIDA (Regulatory Inactivation of DnaA) [12,13,14] and DDAH (datA-dependent DnaAATP hydrolysis) [15] to prevent re-initiation. At later cell cycle stages DnaAADP is reactivated at the two DnaA-Reactivating Sequences (DARS1 and DARS2) to allow for the round of initiation [16, 17]. DARS1 is not regulated by any known pathway [16], while DARS2 activity is modulated by both an IHF- and Fis-dependent pathway [17]
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