# Global analysis of protein degradation reveals instability of diverse regulators in Escherichia coli

## TL;DR

This study identifies many unstable proteins in E. coli, showing how protein degradation helps bacteria adapt to different conditions.

## Contribution

The work introduces a new method combining chemoproteomics, text mining, and machine learning to identify proteolytic regulation in E. coli.

## Key findings

- Proteolysis regulates diverse homeostatic and stress response proteins in E. coli.
- Mutagenesis of PdeH's N-terminal extension disrupted ClpXP recognition and altered biofilm morphology.
- Lon-mediated degradation of FliZ and CsgD suggests proteolysis aids transitions between motility and biofilm states.

## Abstract

The identification of proteins regulated by proteolysis is important for defining homeostatic, developmental, and stress response pathways in bacteria. We present an experimental and computational analysis of protein degradation that reveals protease substrates among nascent proteins in exponential and stationary phase Escherichia coli cells. We validate the active degradation of proteins involved in motility, biofilm development, metabolism, and proteolysis itself. Our work highlights the role of protein degradation in shaping protein abundance dynamics across protein functionalities and physiological states and identifies opportunities for further investigation of this essential aspect of proteostasis.

Regulated protein degradation underlies the timely execution of essential gene expression programs in bacteria. Here, we deployed time-resolved chemoproteomics, text mining of the PubMed and EcoCyc knowledge bases, and machine learning classification to identify proteolytic regulation in exponential and stationary phase Escherichia coli cultures. We experimentally validated the instability of diverse homeostatic and stress response regulators, including the principal cyclic-di-GMP phosphodiesterase PdeH, the N-end rule substrate chaperone ClpS, and all four A-type domain iron–sulfur cluster carriers, IscA, ErpA, NfuA, and SufA. Mutagenesis of the PdeH N-terminal extension abolished ClpXP recognition, thereby impairing stationary phase depletion of PdeH and altering macrocolony biofilm surface morphology. Unstable proteins synthesized in stationary phase such as the morphology regulator BolA, RNA polymerase ω subunit, and the biofilm regulator BssR were implicated in quiescence. Finally, machine learning–assisted substrate identification revealed Lon-mediated degradation of two opposing key regulators of surface adhesion, the RpoS antagonist FliZ and the major biofilm regulator CsgD, suggesting proteolysis may hasten transitions between motility and sessility. Together, these results highlight the role of regulated proteolysis in driving physiological adaptation for this model organism.