The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli

PLoS Genet. 2015 Jun 5;11(6):e1005276. doi: 10.1371/journal.pgen.1005276. eCollection 2015 Jun.

Abstract

The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated. In this work we have performed cell cycle analysis of cells that contain a surplus of ATP-DnaA and asked whether initiation then occurs earlier. It does not. Cells with more than a 50% increase in the concentration of ATP-DnaA showed no changes in timing of replication. We suggest that although ATP-DnaA is the main actor in initiation of replication, its accumulation does not control the time of initiation. ATP-DnaA is the motor that drives the initiation process, but other factors will be required for the exact timing of initiation in response to the cell's environment. We also investigated the in vivo roles of datA dependent DnaA inactivation (DDAH) and the DnaA-binding protein DiaA. Loss of DDAH affected the cell cycle machinery only during slow growth and made it sensitive to the concentration of DiaA protein. The result indicates that compromised cell cycle machines perform in a less robust manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • DNA Replication*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*

Substances

  • Bacterial Proteins
  • Carrier Proteins
  • DNA-Binding Proteins
  • DiaA protein, E coli
  • DnaA protein, Bacteria
  • Adenosine Triphosphate

Grant support

This work was supported by The Norwegian Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.