Document Type

Dissertation

Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Microbiology

First Advisor

Craig D. Ellermeier

Abstract

Extra-Cytoplasmic Function (ECF) σ factors are a subset of σ factors many organisms use to transcribe specific genes in response to environmental cues. In the absence of an inducing signal, ECF σ factors are inhibited by an anti-σ factor that prevents the ECF σ factor from interacting with RNA polymerase. The ECF σ factor σV from Bacillus subtilis is activated in response to lysozyme stress. Lysozyme damages bacterial peptidoglycan by cleaving at the 1,4-ß-linkage between N-acetylmuramic acid and N-acetylglucosamine. In the absence of lysozyme, the activity of σV is inhibited by the anti-σ factor RsiV, a single pass transmembrane protein. The two main components of this project have been to elucidate the mechanism of σV activation and examine how this system senses lysozyme stress.

In chapter 2 we show that the activation of σV is specific to lysozyme, and the anti-σ factor RsiV is degraded by a step wise proteolytic cascade known as Regulated Intramembrane Proteolysis (RIP). In the presence of lysozyme, the extracellular domain of RsiV is removed by cleavage at site-1. Upon removal of the extracellular domain, the site-2 protease, RasP, cleaves RsiV within the membrane. The remainder of RsiV is degraded by cytosolic proteases allowing σV to interact with RNA polymerase. In response to lysozyme stress σV activates an O-actyltransferase, OatA, which modifies the peptidoglycan to prevent further lysozyme damage.

Our studies in chapter 3 identifed the protease(s) responsible for site-1 cleavage of RsiV and revealed RsiV directly interacts with lysozyme. We determined the cleavage site of the site-1 protease using N-terminal sequencing, and demonstrate that disruption of site-1 cleavage blocks σV activation. Site-1 cleavage occurs at what appears to be a signal peptide cleavage site. We demonstrate that four out of the five signal peptidases from B. subtilis are able to cleave RsiV at site-1 in vitro only in the presence of lysozyme. Additionally, we show that the extracellular domain of RsiV directly binds the inducing substrate lysozyme.

In chapter 4 we focus on determining if the interaction between RsiV and lysozyme is necessary for σV activation. Based on the co-crystal structure of RsiV and lysozyme we mutated sveral residues predicted to be involved in binding. One combination of RsiV mutations (S169W, P259A, Y261A) was unable to activate σV and subsequently was unable to bind lysozyme. We propose a RIP dependent mechanism of σV activation that is contingent upon the anti-σ factor (RsiV) directly binding the inducing signal (lysozyme) to present the site-1 cleavage site to signal peptidase.

The co-crystal structure of RsiV and lysozyme also revealed that RsiV interacts with the active site of lysozyme. We demonstrate that purified RsiV inhibits lysozyme activity suggesting RsiV provides an additional lysozyme response mechanism. Thus, the anti-σ factor RsiV senses the presence of lysozyme, activates σV, and protects against further lysozyme damage.

Keywords

ECF sigma Factors, Lysozyme, sigma factors

Pages

xiii, 169 pages

Bibliography

Includes bibliographical references (pages 155-169).

Comments

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Copyright

Copyright © 2015 Jessica Lauren Hastie

Included in

Microbiology Commons

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