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