Genetic and biochemical analysis of the colicin I receptor gene of Escherichia coli: Structural organization and transcriptional control mechanisms
Griggs, David William
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Permalink
https://hdl.handle.net/2142/20009
Description
Title
Genetic and biochemical analysis of the colicin I receptor gene of Escherichia coli: Structural organization and transcriptional control mechanisms
Author(s)
Griggs, David William
Issue Date
1989
Doctoral Committee Chair(s)
Konisky, Jordan
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
Biology, Microbiology
Language
eng
Abstract
The cir gene, which encodes the colicin I receptor protein, is one of several outer membrane proteins synthesized by Escherichia coli in response to iron limitation. The location, organization, and regulation of cir and its neighboring genes were examined by a molecular genetic approach. Following cloning of the cir region, physical mapping and complementation analysis established the position of cir between mgl and nfo on the E. coli chromosome. A gene encoding a 32,000-dalton protein was located downstream of and adjacent to cir, was not part of the same transcriptional unit, and was not regulated by iron. The Cir protein was processed by removal of a signal peptide and the mature form had an amino terminal sequence which closely resembled that of other TonB-dependent proteins.
The molecular basis for iron-regulated expression of cir was investigated by genetic and biochemical means. Deletion analysis of the cloned promoter region allowed delineation of sequences necessary for control of transcription initiating at the two promoters P1 and P2. Gel retardation assays were used to demonstrate a highly specific interaction between purified Fur (ferric uptake regulation) protein and the iron control region. An operator sequence spanning 43-47 base pairs and completely encompassing the two promoters was identified by DNase I protection experiments, with binding occurring in a metal-dependent fashion. Thus, during iron replete growth, Fur appears to act as a repressor of transcription by blocking formation of a DNA-RNA polymerase complex. Expression of cir in an miaA mutant was comparable to that in a wild type strain, so no ancillary role in iron-regulation based on the modification of the affected tRNA species was detected. Characterized and putative Fur recognition sites from several genes were analyzed and classified by statistical methods.
In both high- and low-iron media, functional Crp protein and cyclic adenosine 3$\sp\prime$,5$\sp\prime$-monophosphate (cAMP) were required for maximal production of the receptor. A potential cAMP-CRP binding site located in the long leader region between the cir promoters and the start of the protein-coding region was suggested by sequence and mutational analysis.
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