Identification of a 29 kDa protein in the envelope of Mycobacterium smegmatis as a putative ferri-exochelin receptor

Evidence of a direct association between ferri-exochelin, the major extracellular siderophore of Mycobacterium smegmatis, and a 29 kDa protein has been obtained by three separate methods. (1) Direct binding of ⁵⁵Fe(III)-exochelin by the 29 kDa protein in an envelope preparation from iron-deficient cells was demonstrated by the extraction of a complex with the nondenaturing detergent CHAPS, and subsequent CHAPS-PAGE and autoradiography. (2) Affinity chromatography on a chemically synthesized ferri-exochelin-Sepharose 4B matrix led to the retention of the 29 kDa protein and a 25 kDa protein. The smaller protein was partially eluted with 1 mM ferri-exochelin although it did not form a stable complex with ferri-exochelin. The 29 kDa protein could not be eluted from the affinity matrix with 1 mM ferri-exochelin either alone or with 1 M NaCl. Only 2% (w/v) SDS could do this, but resulted in protein denaturation. (3) Incubation of ⁵⁵Fe-exochelin with CHAPS-solubilized envelope proteins in free solution followed by ion-exchange chromatography resolved three radioactive peaks; subsequent analysis by SDS-PAGE showed that the peak with the highest ⁵⁵Fe-binding activity per unit protein contained both the 29 and 25 kDa proteins. A direct association was demonstrated between the 29 kDa protein and ⁵⁵Fe-exochelin by gel filtration. The evidence suggests that the 29 kDa iron-regulated envelope protein of M. smegmatis is a ferri-exochelin-binding protein and that the 25 kDa protein, which corresponds in size to a previously reported iron-regulated envelope protein in this bacterium, may have a role in the formation or maintenance of this complex. Proteins extracted from the cell envelope of iron-deficient M. smegmatis with CHAPS were dialysed to remove the detergent, incorporated into liposome suspensions and then incubated with ⁵⁵Fe(III)-exochelin. This increased the retention of ⁵⁵Fe by 133-fold compared to proteins not placed in liposomes. Retention of ⁵⁵Fe was dependent upon the protein loading of the liposomes. Gel filtration confirmed that the iron was retained by these vesicles and even after dialysis the majority of ⁵⁵Fe was still retained by the vesicles. Re-solubilization of the labelled proteo-liposomes in various detergents gave limited recovery of a ferri-exochelin-protein complex. Attempts to resolve this complex by Triton X-100 PAGE led to separation of the two entities. The complex was stable, however, in a CHAPS-PAGE system.