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Title:Studies of the structure, evolution, and TonB-dependence of the colicin I receptor of Escherichia coli
Author(s):Nau, Cynthia Dawn
Doctoral Committee Chair(s):Konisky, Jordan
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
Abstract:The colicin I receptor (Cir) is an outer membrane protein, the expression of which is iron regulated. The DNA downstream of the cloned cir gene, when expressed in a maxicell system, encoded a protein of molecular weight 32,000. The expression of this protein was determined to be directed toward the cir gene and uneffected by iron. When the gene was inactivated by insertional mutagenesis, Cir expression and function were unaltered. The gene encoding the 32 kD protein was sequenced. The predicted protein sequence was not significantly related to any of the protein sequences found in the GenBank protein database.
The nucleotide sequence of the cloned cir gene was determined. Several previously noted characteristics of other outer membrane proteins were also identified in the predicted sequence of Cir. These include an overall acidic nature, the absence of long hydrophobic stretches of amino acids, and a lack of predicted $\alpha$-helical structure. The Cir protein was determined to be significantly related to four other TonB-dependent transport proteins. The sequences of these five proteins were multiply aligned, and an evolutionary tree was constructed from the alignment.
Among the regions of homology shared by the TonB-dependent transport proteins is a domain with which the TonB protein putatively interacts. The region of the cir gene which encodes this putative TonB-interaction site was mutagenized in vitro. One of the mutations obtained replaced valine 10 with a glycine. The phenotype of this mutant was identical to the phenotype of a tonB mutant. Cells carrying this mutation were insensitive to colicin I but bound $\sp{125}$I-colicin I at wild-type levels. The phenotype conferred by this mutation was suppressible by mutations in the tonB gene indicating that the Cir and TonB proteins directly interact.
Issue Date:1989
Rights Information:Copyright 1989 Nau, Cynthia Dawn
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9010967
OCLC Identifier:(UMI)AAI9010967

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