# A novel membrane stress response that blocks chromosomal replication by targeting the DnaA initiator via the ClpP protease

## TL;DR

The study reveals a new bacterial stress response where membrane stress blocks DNA replication by degrading a key protein called DnaA through the ClpP protease.

## Contribution

A novel stress response pathway is identified that links membrane stress to replication arrest via ClpP protease and DnaA degradation, independent of Lon and (p)ppGpp.

## Key findings

- Membrane stress activates ClpP protease, which degrades DnaA and blocks DNA replication.
- DnaA loss is sufficient to cause growth arrest under membrane stress.
- The pathway operates independently of Lon protease and (p)ppGpp, distinguishing it from other stress responses.

## Abstract

In Escherichia coli, membrane stress due to interrupted lipoprotein (Lpp) maturation impairs DNA replication and arrests cell growth. How Lpp maturation and DNA replication are connected remains unclear. We demonstrate that upon membrane stress, the Rcs stress-response pathway is activated, and the replication initiator DnaA is lost, which explains the replication block. However, Lon protease, a key regulator of the Rcs pathway, is not required for the DnaA loss. We further ruled out the involvement of (p)ppGpp, one of the major mediators of stress responses in bacteria. On the other hand, upon deletion of the ClpP protease gene, DnaA was stable, replication was not inhibited, and there was no cell-growth arrest. In wild-type cells, overexpression of DnaA was lethal even without the membrane stress apparently from hyperinitiation. In ∆crp cells, hyperinitiation was restrained, and overexpression of DnaA was able to overcome the growth arrest. ∆fis cells, which were earlier found resistant to the membrane stress, showed DnaA stability and normal replication upon stress-inducing treatments. We conclude that DnaA loss suffices to explain the growth arrest upon the membrane stress. The stress-response pathway described here appears novel because of its independence from Lon and (p)ppGpp, which have been implicated in other stress responses that block DNA replication.

The observation that DNA replication stress can block cell division in E. coli (SOS response) introduced the concept of checkpoint control in the cell cycle. Here, we describe a novel checkpoint control that functions in the opposite direction: membrane stress causing replication block. We show how the accumulation of precursor lipoprotein (pLpp) could block replication. The pLpp accumulation causes a response culminating in activating the ClpP protease that blocks replication by targeting the initiator DnaA. DnaA being vital and highly conserved, a detailed understanding of the response pathway is likely to open new avenues to treat bacterial infection.