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DNA replication initiation in Bacillus subtilis: structural and functional characterization of the essential DnaA-DnaD interaction.

ABSTRACT: The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

SUBMITTER: Martin E

PROVIDER: S-EPMC6393240 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Project description:Bacillus subtilis and Escherichia coli are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, the two organisms follow the same phenomenological principle, known as the adder principle, for cell size homeostasis. We thus asked to what extent B. subtilis and E. coli share common physiological principles in coordinating growth and the cell cycle. We measured physiological parameters of B. subtilis under various steady-state growth conditions with and without translation inhibition at both the population and single-cell levels. These experiments revealed core physiological principles shared between B. subtilis and E. coli Specifically, both organisms maintain an invariant cell size per replication origin at initiation, under all steady-state conditions, and even during nutrient shifts at the single-cell level. Furthermore, the two organisms also inherit the same "hierarchy" of physiological parameters. On the basis of these findings, we suggest that the basic principles of coordination between growth and the cell cycle in bacteria may have been established early in evolutionary history.IMPORTANCE High-throughput, quantitative approaches have enabled the discovery of fundamental principles describing bacterial physiology. These principles provide a foundation for predicting the behavior of biological systems, a widely held aspiration. However, these approaches are often exclusively applied to the best-known model organism, E. coli In this report, we investigate to what extent quantitative principles discovered in Gram-negative E. coli are applicable to Gram-positive B. subtilis We found that these two extremely divergent bacterial species employ deeply similar strategies in order to coordinate growth, cell size, and the cell cycle. These similarities mean that the quantitative physiological principles described here can likely provide a beachhead for others who wish to understand additional, less-studied prokaryotes.

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DNA replication initiation in Bacillus subtilis: structural and functional characterization of the essential DnaA-DnaD interaction.

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