Studies in the iron uptake mechanism of Mycobacterium smegmatis : identification of components of the iron uptake system

Dover, Lynn G.

Abstract

The research in this thesis is concerned with the identification of the envelope proteins involved in the uptake of ferri-exochelin MS by M. smegmatis. Analysis of envelope protein profiles under iron sufficiency and iron deficiency identified a family of seven proteins with increased expression under iron-limiting conditions. The results of this study were in broad agreement with earlier observations by other workers. The expression of these seven iron-regulated envelope proteins (IREPs) was not increased on heat shock treatment or on zinc limitation demonstrating that their elaboration was a specific response to iron deficiency. The 29 kDa envelope protein suggested as a putative receptor protein by earlier studies, however, was found to be constitutively expressed at a high level irrespective of the iron status of the culture in our hands.

The majority of the work in this thesis is concerned with the isolation of a ferri-exochelin MS receptor protein. A suite of techniques was developed to identify a receptor via the formation of ferri-exochelin MS-IREP complexes.

The 29 kDa IREP was implicated as the major ferri-exochelin MS-binding protein (PEBP) of M. smegma tis by every protocol attempted:

i. a radio-labelled ferri-exochelin-29 kDa complex was formed in situ in a crude envelope preparation, extracted and separated by electrophoresis.
ii. a 29 kDa protein was purified from crude envelope extracts of the bacterium to homogeneity by affinity chromatography on ferri-exochelin MS-Sepharose.
iii. a 55Pe-labelled-29 kDa ferri-exochelin was isolated from a crude envelope extract by anion-exchange chromatography and gel filtration chromatography.

Anion exchange and affinity chromatography fractionations also provided evidence that a 25 kDa !REP may be involved in the ferri-exochelin uptake mechanism as a component of a receptor complex along with the 29 kDa FEBP.

These techniques were hindered, however, by a lack of sensitivity. In order to address this problem extracted envelope proteins were incorporated into a simple liposome preparation. The ferri-exochelin MS-binding activity of the resulting proteoliposome suspension was 133-fold greater than that exhibited by extracted envelope proteins in a buffered detergent solution. This activity was protein-dependent and irreversible on dialysis suggesting a stable binding event, rather than a non-specific association, with the proteo-liposome had occurred. A stable 55 Fe-labelled ferriexochelin-protein complex was extracted from the proteo-liposomes and isolated by gel filtration chromatography. A molecular size of 57 ± 4 kDa was estimated for the complex but the size of its components could not be reliably determined due to their abnormal electrophoresis on SDS-PAGE. The estimated size of this complex was consistent with the proposed hypothesis that a 29 : 25 kDa heterodimer acts as the ferriexochelin receptor of M. smegmatis.

Citation

Dover, L. G. (1995). Studies in the iron uptake mechanism of Mycobacterium smegmatis : identification of components of the iron uptake system. (Thesis). University of Hull. Retrieved from https://hull-repository.worktribe.com/output/4218720

• Thesis Type: Thesis
• Deposit Date: Nov 24, 2016
• Publicly Available Date: Feb 23, 2023
• Keywords: Applied biology
• Public URL: https://hull-repository.worktribe.com/output/4218720
• Additional Information: Department of Applied Biology, The University of Hull
• Award Date: Apr 1, 1995