Growth and cytochrome synthesis in a hemin-requiring mutant of Spirillum itersonii.

Spirillum itersonii forms considerable amounts of band c-type cytochrome when grown under low aeration, and we have suggested that synthesis of the heme prosthetic group, derived from glycine and succinyl CoA, might be important in regulating the formation of the complete hemoproteins (1). The isolation of a mutant strain requiring hemin (ferric protoporphyrin chloride) has enabled a more direct study of the influence of the prosthetic group on cytochrome synthesis. The parent strain and microbiological and analytical procedures were as described previously (1). The basal medium was glutamate-succinateglycine plus 0.1% yeast extract and 0.1% Tween-80 to keep hemin in solution; the stock solution of hemin (2 mM), in 0.01 N NaOH and 50% ethyl alcohol, was added aseptically. The mutant H27/8 was isolated after mutagenesis with N-methyl-N-nitroso-N'-nitroguanidine; treated cells were plated on basal medium containing 0.1 mM 6-aminolevulinate (ALA) and 0.005 mm hemin. Colonies were tested individually for growth with and without these supplements. Mutant H27/8 responded to hemin or to ALA, although the latter was less effective (Fig. 1); 0.005 mM protoporphyrin and magnesium protoporphyrin were inactive. ALA synthase activity was not detectable in extracts of the mutant, and the block presumably was at this stage. Hemin was needed for maximum cytochrome synthesis, and only very low levels were found in ALA-grown cells (Table 1). The respiratory rates with glutamate as substrate were, respectively, 4 and 17 ,umoles of 02 per hr per mg of protein for cells grown with 0.1 mm ALA or 0.005 mM hemin. Both band c-type cytochromes were formed by hemin-grown cells, and the response to aeration was similar to the wild-type (Table 1). Under low aeration, cytochrome synthesis was enhanced, cytochrome c being the predominant pigment. High aeration repressed cytochrome synthesis at all concentrations of hemin tested. This does not support the idea that oxygen represses hemoprotein synthesis by favoring succinate utilization via the Krebs cycle and thereby limiting its availability for heme synthesis. In vitro experiments have suggested that the iron of heme c may be incorporated after combination of the protein moiety with protoporphyrinogen; this mechanism would preclude iron protoporphyrin as an intermediate (3). However, experiments with 59Fe-hemin, prepared as de-