Abstract

DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

Highlights

  • Faithful chromosomal replication requires regulatory networks that ensure the precise coordination of DNA replication with other cell cycle events

  • Our previous discovery of the HdaA protein [16], which is homologous to the E. coli Hda protein, suggested that Regulatory Inactivation of DnaA (RIDA) may occur in C. crescentus, and that this mechanism may restrict the initiation of chromosomal replication to only once per cell cycle in this particular bacterial species

  • We showed that the DnaA(R357A) mutant protein in C. crescentus retains its ability to promote the initiation of chromosomal replication in vivo, and is even hyper-active as an initiator compared to the wild-type DnaA protein, as indicated by the severe over-replication phenotype of cells that over-express DnaA(R357A) (Fig.4)

Read more

Summary

Introduction

Faithful chromosomal replication requires regulatory networks that ensure the precise coordination of DNA replication with other cell cycle events. Binding of ATP to these proteins is thought to induce conformational changes that activate these proteins to promote the initiation of DNA replication at the correct time of the cell cycle. Besides its function as an initiator of bacterial chromosome replication, DnaA is a transcription factor that binds to many promoters to regulate their activities in diverse bacterial species [3,4]. It is still unclear whether oscillations in the levels of DnaA-ATP influence the timing of gene transcription in most cases [5,6]

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call