Auxotrophic mutants of the filamentous cyanobacterium Anabaena variabilis were isolated by a method in which, after mutagenesis and before penicllin enrichment, mutant and wild-type cells were separated by cavitation. Auxotrophs were identified by their inability to grow on minimal medium, and they were partially characterized by replica plating to media supplemented with single nutrients or specific groups of nutrients. Of the 83 auxotrophs isolated, 65 required an inorganic source of nitrogen for growth. In addition, auxotrophs were isolated that required methionine (six), uracil (two), adenine (one), biotin (two), and nicotinic acid (two). (The number of isolates of each type is indicated in parentheses.) The nutrient requirements of five auxotrophs appeared complex and were not determined. A large proportion of the mutants requiring inorgainic fixed nitrogen was altered in the differentiation of heterocysts. The following morphological aberrancies were observed: abnormally high and abnormally low frequencies of heterocysts; thick, uneven heterocyst envelopes; incompletely developed pore regions; very distinct pore regions; and protoplasts separated from the envelope of the heterocyst. Spontaneously occurring, N2-fixing, prototrophic revertants of mutants with aberrant heterocysts have been isolated at a frequency of 2 X 10(-8) to 4 X 10(-8) of the cells plated. That most such revertants produced morphologically normal heterocysts is consisten with the idea that heterocysts play an essential role in aerobic N2 fixation.

Fructose is specifically taken up by nitrogen-fixing cultures of Anabaena variabilis in the light and lowers the doubling time from 24 to 8 h. The kinetics for both fructose-dependent growth and fructose uptake are exponential. The apparent Km for fructose uptake in N2-fixing cultures is 160 ,IM for cells not previously exposed to fructose and 50,uM in cells adapted to fructose. Picomolar amounts of [14C]fructose are scavenged from the medium and accumulate in filaments. Heterocysts of fructose-adapted filaments accumulate 14C from fructose within 20 min. Short-term experiments with fructose-starved cultures provide evidence that nitrogenase activity, protein, and chlorophyll content change within one generation time upon addition of fructose. In long-term experiments, the amount of fructose initially present in the medium determines heterocyst number and packed-cell volume. Photosynthetic oxygen evolution and amounts of chlorophyll decrease with exogenous fructose concentrations greater than 20 mM.

Mutants of Anabaena variabilis deficient in the envelope glycolipids of heterocysts have no or very low nitrogenase activity when assayed aerobically. Revertants capable of aerobic growth on N2 have increased quantities of these glycolipids. Among mutants which require fixed nitrogen for growth in air and which have a normal complement of glycolipids, one expresses high nitrogenase activity at low oxygen tension. Three others show high nitrogenase activity only in the presence of dithionite and are therefore impaired in electron transfer.

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