IHF and Fis as Escherichia coli Cell Cycle Regulators: Activation of the Replication Origin oriC and the Regulatory Cycle of the DnaA Initiator

Abstract

This review summarizes current knowledge about the mechanisms of timely binding and dissociation of two nucleoid proteins, IHF and Fis, which play fundamental roles in the initiation of chromosomal DNA replication in Escherichia coli. Replication is initiated from a unique replication origin called oriC and is tightly regulated so that it occurs only once per cell cycle. The timing of replication initiation at oriC is rigidly controlled by the timely binding of the initiator protein DnaA and IHF to oriC. The first part of this review presents up-to-date knowledge about the timely stabilization of oriC-IHF binding at oriC during replication initiation. Recent advances in our understanding of the genome-wide profile of cell cycle-coordinated IHF binding have revealed the oriC-specific stabilization of IHF binding by ATP-DnaA oligomers at oriC and by an initiation-specific IHF binding consensus sequence at oriC. The second part of this review summarizes the mechanism of the timely regulation of DnaA activity via the chromosomal loci DARS2 (DnaA-reactivating sequence 2) and datA. The timing of replication initiation at oriC is controlled predominantly by the phosphorylated form of the adenosine nucleotide bound to DnaA, i.e., ATP-DnaA, but not ADP-ADP, is competent for initiation. Before initiation, DARS2 increases the level of ATP-DnaA by stimulating the exchange of ADP for ATP on DnaA. This DARS2 function is activated by the site-specific and timely binding of both IHF and Fis within DARS2. After initiation, another chromosomal locus, datA, which inactivates ATP-DnaA by stimulating ATP hydrolysis, is activated by the timely binding of IHF. A recent study has shown that ATP-DnaA oligomers formed at DARS2-Fis binding sites competitively dissociate Fis via negative feedback, whereas IHF regulation at DARS2 and datA still remains to be investigated. This review summarizes the current knowledge about the specific role of IHF and Fis in the regulation of replication initiation and proposes a mechanism for the regulation of timely IHF binding and dissociation at DARS2 and datA.

Genetic components of Escherichia coli involved in its complex prey-predator interaction with Myxococcus xanthus

“…Subsequent investigation revealed the pivotal roles played by the dusB and fis genes in driving this observed phenotype. To the best of our knowledge, this represents the initial report detailing the involvement of the global regulator encoded by the dusB-fis operon ( Crozat et al, 2010 ; Gerganova et al, 2015 ; Kasho et al, 2023 ) in the regulation of prey susceptibility in E. coli .…”

“…Through literature review, we have noticed that in E. coli , the dusB - fis operon encodes the global regulator Fis (factor for inversion stimulation), an abundant nucleoid-associated protein (NAP) that undergoes transient expression during the early exponential phase, which serves as an important global regulator of cellular metabolism, coordinating chromosomal DNA topology and ribosomal biosynthesis ( Crozat et al, 2010 ; Gerganova et al, 2015 ; Kasho et al, 2023 ). Given that the dusB-fis operon regulates a multitude of crucial physiological processes in E. coli , establishing a direct causal link between the mutation of the dusB-fis operon and the observed defensive phenotype in predation through bioinformatics analysis becomes challenging.…”

“…Fis is a highly abundant small DNA-binding protein within E. coli , playing a multifaceted role in regulating various biological processes, including transcription, recombination, replication reaction, nucleoid compaction, and chromosome conformation ( Crozat et al, 2010 ; Nafissi et al, 2012 ; Duprey et al, 2014 ; Kasho et al, 2023 ). The expression profile of Fis is characterized by elevated levels during rapid growth under nutrient-rich conditions, while it diminishes during slow growth in nutrient-poor environments or in the stationary phase ( Ali Azam et al, 1999 ).…”

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

“…In summary, here we have measured oscillations in DnaA activity and SeqA repression that integrate the effects from several regulatory mechanisms to regulate the initiation of DNA replication. Given the essential role of DnaA in cell growth and adaptation, these processes regulate its activity to maintain a stable DNA replication program across growth conditions ( 58 – 60 ).…”

Read-through transcription of tRNA underlies the cell cycle-dependent dissociation of IHF from the DnaA-inactivating sequence datA

“…The E. coli chromosome is a circular double-strand 4.6 Mb DNA molecule that forms a compact structure, the nucleoid, condensed by binding of various nucleoid-associated proteins (NAPs) and a DNA superstructure, such as supercoiled DNA ( Dillon and Dorman, 2010 ; Hołówka and Zakrzewska-Czerwińska, 2020 ; Molan and Žgur Bertok, 2022 ). The E. coli chromosome has a single replication origin, oriC (located at 84.6 min of the genome map), and initiation of replication at oriC is regulated precisely during the cell cycle so that, even in rapidly growing cells, initiation occurs simultaneously at multiple sister oriCs ( Frimodt-Møller et al, 2017 ; Hansen and Atlung, 2018 ; Grimwade and Leonard, 2021 ; Kasho et al, 2023 ). In E. coli , the initiation reactions of DNA replication occur in a higher-order complex that includes oriC , the initiator protein DnaA, and a NAP IHF (integration host factor).…”