The dissociable promoter recognition subunit RpoS (also known as ?s) is the master transcriptional regulator of the general stress response in ?-proteobacteria, and plays key roles in the virulence of many pathogens, including human, plant and animal pathogens. Under certain conditions, such as at the transition from the logarithmic to the stationary phase or in the presence of stress signals, RpoS redirects the core RNA polymerase machinery to a subset of promoters to reprogram transcription. However, intracellular RpoS levels are not steady ? they are low in actively dividing cells, and substantially increased upon entering the stationary phase or upon encountering stress. To achieve proper regulation, there is tight control over RpoS levels, with the major point of regulation occurring at the level of RpoS proteolysis by the ATP-dependent ClpXP machine. Our central focus is to understand the mechanisms of RpoS proteolysis by ClpXP as well as its regulation by an emerging family of proteins collectively called anti-adaptors. In order to be degraded, RpoS is presented to ClpXP by a unique, highly specific adaptor called RssB, which acts catalytically, without being degraded. In turn, RssB itself is regulated by interactions with stress-specific anti-adaptors. Our work will focus on the structure and function of three-anti-adaptors: IraD (induced by oxidative stress and DNA damage), IraM (induced by magnesium starvation) and IraP (induced by phosphate starvation). These anti-adaptors share no sequence homology, and only weak homology with protein of known structure, warranting a structural biology effort aimed at deciphering the underlying mechanisms of RssB recognition. We will determine the structures of IraD, IraM and IraP, both in isolation and bound to RssB using X-ray crystallography. This will allow us to pinpoint, at atomic resolution, residues important for anti-adaptor/RssB interactions, and also regulation of the anti-adaptors themselves by oligomerization. We will complement these structural studies with molecular genetics, microscopy and functional assays for protein-protein interactions and RpoS degradation, which will allow us to correlate in vitro behavior with in vivo observations. We will also determine structures of a RpoS- RssB-ClpXP assembly using cutting-edge methods in electron cryo-microscopy, which will allow us to understand the RpoS and RssB conformational dynamics at the core of this paradigmatic mode of regulated proteolysis in bacteria. Overall, this work will not only bring fundamental, mechanistic insights, but will also open the way to the development of novel antibacterials that could target ClpXP directly, or, adaptor/anti- adaptor interfaces. The RpoS regulon has been reported to comprise up to 10% of the Escherichia coli genome, and RpoS itself plays important roles in bacterial persistence, host-pathogen interactions and biofilm formation, which underlie 80% of all infections.

Public Health Relevance

In the wild, not under laboratory conditions, nutrients are limiting and, generally, bacteria do not actively divide but are found in the stationary phase. RpoS is the master regulator of the general stress response and of the stationary phase and it plays crucial roles in the virulence and persistence of many human, animal and plant pathogens. Understanding RpoS regulation can thus provide key insights into the development of antimicrobials to combat infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM121975-03
Application #
9657045
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Ainsztein, Alexandra M
Project Start
2017-04-01
Project End
2022-03-31
Budget Start
2019-04-01
Budget End
2020-03-31
Support Year
3
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
Le, Tung T; Yang, Yi; Tan, Chuang et al. (2018) Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism. Cell 172:344-357.e15
Vemu, Annapurna; Szczesna, Ewa; Zehr, Elena A et al. (2018) Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation. Science 361: