Direct single-cell observation of a key Escherichia coli cell-cycle oscillator

• DOI: 10.1126/sciadv.ado5398
• Publication Date: 2024-07-17T18:00:47Z
• Authors: Ilaria Iuliani; Gladys Mbemba; Marco Cosentino L...; Bianca Sclavi
• Keywords: DNA-Binding Proteins; DNA Replication; Bacterial Proteins; Escherichia coli Proteins; Cell Cycle; Escherichia coli; Biomedicine and Life Sciences; Gene Expression Regulation, Bacterial; Single-Cell Analysis; Promoter Regions, Genetic; Bacterial Outer Membrane Proteins

Initiation of DNA replication in Escherichia coli is coupled to cell size via the DnaA protein, whose activity is dependent on its nucleotide-bound state. However, the oscillations in DnaA activity have never been observed at the single-cell level. By measuring the volume-specific production rate of a reporter protein under control of a DnaA-regulated promoter, we could distinguish two distinct cell-cycle oscillators. The first, driven by both DnaA activity and SeqA repression, shows a causal relationship with cell size and divisions, similarly to initiation events. The second one, a reporter of DnaA activity alone, loses the synchrony and causality properties. Our results show that transient inhibition of gene expression by SeqA keeps the oscillation of volume-sensing DnaA activity in phase with the subsequent division event and suggest that DnaA activity peaks do not correspond directly to initiation events.