Date of Award

August 2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Sonia L. Bardy

Committee Members

Mark McBride, Steven Forst, Ching-Hong Yang, Gyaneshwar Prasad

Abstract

Bacteria detect environmental signals using membrane-bound methyl-accepting chemotaxis proteins (MCPs), which are part of a larger complex of chemosensory proteins. Pseudomonas aeruginosa has four functionally distinct chemosensory protein complexes. The Chp chemosensory system regulates type IV pili mediated twitching motility and intracellular levels of cAMP by modulating the activity of an adenylate cyclase, CyaB. The Chp system is also proposed to be involved in type IV pili mediated directional twitching motility towards phosphatidylethanolamine (PE). PilJ is the only MCP predicted to be associated with the Chp system. In this study we investigated different domains of PilJ in order to decipher their roles in signal transduction and localization of the Chp chemosensory system.

Our results show that both the periplasmic and transmembrane domains are involved in signal transduction. A PilJ periplasmic domain mutant (PilJΔ74-273) showed a partial but significant decrease in cAMP levels highlighting the importance of this domain in regulation of this phenotype. However, when sufficient intracellular cAMP is provided, twitching motility occurs in the presence or absence of PE independent of the periplasmic domain. To maintain the inner membrane localization while assaying the role of the transmembrane domains of PilJ, Tsr-PilJ chimeric proteins were used. These fusion proteins showed impaired signal transduction. Through localization studies, we discovered that the role of the PilJ transmembrane domains is not limited to signal transduction. Using fluorescence microscopy, an examination of the intracellular localization of C-terminus truncations of PilJ revealed that the second transmembrane domain plays a role in polar localization of PilJ. This is the first report where the localization of a MCP is impacted by a domain other than the cytoplasmic domain. Determining the internal localization cues for this MCP was important as MCPs are reported to be instrumental in chemosensory cluster formation and localization. Indeed, PilJ is needed for PilI foci formation as demonstrated by fluorescence microscopy studies. Taken together, these studies suggest that the PilJ transmembrane domains are important for both signal transduction and localization. Establishing the importance of PilJ for the potential nucleation of the Chp system paves the path for future work to identify potential partners that would directly or indirectly control the polar localization of PilJ.

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