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Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0.02 microgram Fe ml-1) and iron-sufficient (2.0 to 4.0 micrograms Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins: the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.

In mycobacterial growth medium 40 to 400 ,uM citrate was required to solubilize 2 FM "Fe. This solubiized "5Fe was taken up into both iron-deficient and ironsufficient washed cell suspensions of Mycobacterium smegmatis and Mycobacterium bovis BCG. Although the 55Fe was taken up into the cell, the citrate was not. The uptake system with M. smegmatis was not inhibited by electron transport inhibitors, uncouplers of oxidative phosphorylation, or thiol reagents and was saturable with iron at approximately 35 ,uM. The system was independent of the iron transport systems already known to exist in M. smegmatis: i.e., the two exochelin routes of assimilation as well as the mycobactin-salicylate system. It was not induced by the presence of 400 ,IM citrate in the growth medium, nor did the presence of citrate in the medium affect the production of either exochelin or mycobactin.

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