Heme biosynthetic activity in the symbiotic association involving crithidial flagellates and intracellular bacteroids was studied by enzymic, nutritional, and isotope incorporation experiments. Component organisms and their complexes in this association were analyzed separately to determine the underlying cause of the hemin requirement of hemoflagellates and the role of symbiotes in sparing this requirement of two crithidial species. Nutritional study of symbiote-free flagellates showed that their growth requires at least 0.1 mug/ml of hemin, which can be substituted by protoporphyrin IX, but not by the porphyrin precursors, delta-amino-levulinic acid or porphobilinogen. These flagellates, in the presence of protoporphyrin IX, incorporated 59Fe into heme, indicating that they possess ferrochelatase (EC 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, which catalyzes the insertion of iron into protoporphyrin IX. In symbiote-containing flagellates serially cultured in a defined medium free of tetrapyrrole compounds, heme and porphyrins can be detected by a fluorophotometric method, indicative of heme biosynthesis. Study of [14C]glycine incorporation into heme showed that the rate is much higher in symbiote-containing flagellates than in those without symbiotes. Microassay of uroporphyrinogen I synthase [EC 4.3.1.8; porphobilinogen ammonia-lyase (polymerizing)] revealed that the specific activity is high in symbiote-containing flagellates and higher still in isolated symbiotes, but essentially negligible in symbiote-free organisms. It is concluded that the bacterial symbiotes augment a very limited heme biosynthetic capacity of host flagellates by supplying uroporphyrinogen I synthase and perhaps other enzymes preceding ferrochelatase in the heme biosynthetic chain.
