Investigating the role of the three N-terminal HAMP domains of the Aer2 chemoreceptor from Pseudomonas aeruginosa
Pseudomonas aeruginosa is a ubiquitous environmental bacterium and opportunistic human pathogen. It readily adapts to changing environmental conditions with the aid of 26 chemoreceptors that signal to four distinct chemosensory systems. The Aer2 chemoreceptor from P. aeruginosa is an O2 sensor implicated in stress responses, virulence and fine-tuning the chemotaxis (Che) system. Aer2 is the sole chemoreceptor of the Che2 chemosensory system, which forms complexes at the cell pole during stationary phase. Aer2 is soluble, yet membrane-associated and displays an unusual arrangement with its PAS sensing domain sandwiched between three N-terminal and two C-terminal HAMP domains. HAMP domains are widespread signaling modules, typically found as single domains or in poly-HAMP chains in transmembrane and soluble proteins. HAMP domains 1-3 of Aer2 form a poly-HAMP chain that resides outside the signal input-output pathway of Aer2. To understand the role of HAMP1-3 in the context of full-length Aer2, I evaluated HAMP1-3 through sitedirected and deletion mutagenesis, behavioral assays and fluorescence microscopy. My data indicate that HAMP1-3 influence PAS signaling over a
considerable distance. The majority of HAMP mutations, and deletions of helical phase stutters, led to nonresponsive signal-off or off-biased receptors. Introducing PAS signal-on lesions to imitate activated Aer2 also failed to override the effects of HAMP truncations and signal-off lesions. My data demonstrate that HAMP1-3 are critical facilitators of PAS signaling that function as a cohesive, moveable unit to orient and poise PAS dimers for O2-mediated signaling in Aer2. My data also demonstrate that HAMP1, but not its charge, drives the clustering and polar localization of Aer2. Moreover, localization was not contingent on Aer2 function, as HAMP1 signal-off mutants still localized and formed Aer2-dependent clusters. The work presented in this dissertation clarifies the function of HAMP1-3, which lay upstream of the PAS sensing domain, and demonstrates two newly recognized roles for HAMP. Specifically, I have shown that HAMP domains can function as both polar clustering determinants and as facilitators of PAS signaling. Overall, this work provides a deeper understanding of the roles of poly- HAMP chains, and may be applicable to other proteins with non-signaltransducing HAMP domains and poly-HAMP chains.
School of Medicine
Kylie J. Watts
Mark S. Johnson
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Pseudomonas aeruginosa; Methyl-Accepting Chemotaxis Proteins; Histidine Kinase; Adenylyl Cyclases
xvii, 131 p.
Loma Linda University Libraries
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Anaya, Selina, "Investigating the role of the three N-terminal HAMP domains of the Aer2 chemoreceptor from Pseudomonas aeruginosa" (2022). Loma Linda University Electronic Theses, Dissertations & Projects. 1742.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives