Abstract
The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator DnaA-oriC complex under specific growth conditions.
Highlights
Chromosomal replication initiation is tightly regulated to allow cell cycle progression, and its regulation is one of the important targets of stress responses (Sclafani and Holzen, 2007; O’Donnell et al, 2013)
The increased yfdQRST copy numbers inhibited the overinitiation in dnaAcos and seqA mutants, as well as appropriate initiation in wildtype cells
Increasing the copies of yfdQRST led to inhibited colony formation of temperature-sensitive dnaA cells with a mutation in DnaA domain IV, at 30◦C
Summary
Chromosomal replication initiation is tightly regulated to allow cell cycle progression, and its regulation is one of the important targets of stress responses (Sclafani and Holzen, 2007; O’Donnell et al, 2013). Activities of several proteins responsible for the initiation are controlled during the cell cycle (Katayama et al, 2010; Leonard and Grimwade, 2015; Marczynski et al, 2015; Wolanski et al, 2015). In Escherichia coli, the initiator protein DnaA recognizes the chromosomal replication origin, oriC (Ozaki and Katayama, 2009; Kaguni, 2011; Leonard and Grimwade, 2011; Costa et al, 2013; Saxena et al, 2013). After oriC unwinding, DnaB helicase is loaded onto the resultant single-stranded DNA (ssDNA) via interactions with the oriC-bound DnaA multimers and DnaC helicase loader (Kaguni, 2011). After Okazaki fragment synthesis on the lagging strand, the clamp subunit dissociates from DNA polymerase III and remains temporarily loaded on the lagging strand (Langston et al, 2009; Su’etsugu and Errington, 2011; Moolman et al, 2014)
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