A Mutational Analysis of XcpQ, the Outer Membrane Component of the Type II Protein Secretion System From Pseudomonas Aeruginosa
Miyagi, Heather Yukiye
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https://hdl.handle.net/2142/86762
Description
Title
A Mutational Analysis of XcpQ, the Outer Membrane Component of the Type II Protein Secretion System From Pseudomonas Aeruginosa
Author(s)
Miyagi, Heather Yukiye
Issue Date
2000
Doctoral Committee Chair(s)
David N. Nunn
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
The virulence of opportunistic pathogen Pseudomonas aeruginosa is largely dependent on its ability to secrete a number of hydrolytic enzymes and cytotoxins. Lipase, phospholipase C, alkaline phosphatase, elastase, exotoxin A, LasA protease, and LipC cryptic lipase are exported via a Type II secretion apparatus. XcpQ is the sole outer membrane protein component of this system and is a member of the superfamily of proteins called secretins. XcpQ forms extremely stable pore-like outer membrane multimers, through which Type II secreted proteins are thought to pass. In this thesis I present a mutational analysis of the role of XcpQ in Type II protein secretion. A series of XcpQ mutants with single amino acid substitutions were identified by a multilevel screening process designed to isolate mutants with minimal function but near wild type protein stability. These mutants were divided into several classes based on their secretion defects, ability to form stable XcpQ multimers, ability to interfere with a non-mutant secretion apparatus, and phenotypes in an interallelic complementation experiment. The results described in this work allow a refinement of the current model of XcpQ functional domain structure and provide genetic evidence that divides the assembly and function of the Type II secretion apparatus into several steps. Stable monomeric XcpQ mutants resulting from a single amino acid change were isolated for the first time during this study. Phenotypic analysis of monomeric and multimerization-competent mutants show that the xcpQ open reading frame can be divided into two amino-terminal domains required for function in secretion, while the predicted membrane spanning segments in the carboxy-terminus are required for assembly and/or stability of the outer membrane multimer. Analysis of mutant phenotypes show that the assembly of the XcpQ outer membrane multimer can be divided into two steps, and interaction of the assembled XcpQ multimer with the rest of the secretion apparatus can be shown to occur either in two steps or in a single step that requires two loci within the amino-terminus of XcpQ. Substrate secretion may constitute a separate step after apparatus assembly is complete, or may be linked to one or more assembly steps.
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