Directed evolution reveals the mechanism of HitRS signaling transduction in Bacillus anthracis.

Hualiang Pi,Hannah K Lin,Casey J Latario,Devin L Stauff,Gideon H Hillebrand,Jared D Reppart,Eric P Skaar,Samuel J Ivan,Sophia M Carlin,Michelle L Chu,Allen M Toth,Theresa M Koehler

doi:10.1371/journal.ppat.1009148

Hualiang Pi, Hannah K Lin + Show 10 more

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https://doi.org/10.1371/journal.ppat.1009148

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Abstract

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.

Highlights

B. anthracis is a Gram-positive, spore-forming, facultative aerobe, and the causative agent of anthrax
Through unbiased genetic selections and rigorous biochemical analysis, we provide a detailed characterization and structure-function analysis of an entire Two component systems (TCSs) and extend our understanding of the molecular basis of signal transduction through TCSs
The described genetic selection strategies are applicable to any TCS, providing a powerful tool for researchers interested in microbial signal transduction

Summary

Introduction

B. anthracis is a Gram-positive, spore-forming, facultative aerobe, and the causative agent of anthrax. B. anthracis spores can survive extreme temperatures, harsh chemical assaults, and nutrient-poor environments for many years [1]. This pathogen is one of the few infectious agents that have been proven effective as a weapon of bioterror. To survive interactions with the host immune system during infection, B. anthracis has developed comprehensive systems for stress detection and detoxification [3]. This pathogen is an excellent model to study microbial stress responses

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