Mutants of azotobacter that do not fix nitrogen.

Stable strains of mutants of Azotobacter that do not fix nitrogen are difficult to obtain by traditional procedures (Karlsson and Barker, 1948; Lindstrom, 1948). Stumbo and Gainey (1938) demonstrated that culturing Azotobacter on a high nitrate medium resulted in strains that failed to fix nitrogen even after continued transfer in the absence of fixed nitrogen. However, since these strains were not available, we have employed the following modifications of usual procedures to secure a number of mutant strains: (1) Nitrogen mustard or ultraviolet treatment of the cells or the broth, or both, was employed singly and in combinations. (2) Azotobacter cells in the log phase of growth, when stained by the method of Robinow (1942), showed 2 to 4 nuclear bodies per cell. Therefore the culture was permitted to grow in liquid media for some time after the application of the mutagenic agent before plating to obtain isolates. This would permit segregation of any mutant nuclei. (3) This enrichment medium was incubated under an atmosphere of oxygen and hydrogen. The purpose of such incubation was as follows: (a) Hydrogen gas, a competitive inhibitor of nitrogen fixation, will suppress any concurrent fixation of nitrogen in the subculture medium and thus the nonfixing mutants will not be at a selective disadvantage as compared to the normal cells. (b) When Kohn and Harris (1942) grew Ewcherichia coli on a complete medium in the presence of the competitive inhibitor, sulfanilamide, the predominant part of the resulting population became methionine-requiring mutants. Since p-aminobenzoic acid functions in methionine synthesis and the enzyme involved is lost when the culture is grown in the presence of methionine and the inhibitor, the growth of Azotobacter in the presence of hydrogen and fixed nitrogen is somewhat analo-