Production of biochemical mutations in Penicillium.

THE PRODUCTION of strains of Neurospora crassa characterized by a loss of biosynthetic ability is well known, and such strains have contributed materially to our knowledge of biosynthetic processes and their genetic control (see reviews, Beadle, 1945; Bonner, in press). During the course of attempts to induce mutations in Penicillium which might yield strains with higher penicillin yields, a number of strains have been obtained having nutritional requirements more complex than those of the parent strain. Penicillium notatum is an imperfect fungus, and it is therefore impossible to demonstrate the genetic basis of induced biochemical deficiencies. However, several of these strains are interesting from a biochemical standpoint, and probably many of them will prove useful in studies of biosynthesis and of comparative biochemistry. This paper deals with the methods of producing and detecting strainis of Penicillium characterized by nutritional deficiencies, and records the strains that have been obtained. STOCKS AND MEDIA.-Stocks of two species were used in this work, P. notatum and the closely related species P. chrrysogenum. Table 1 lists the several strains used and their interrelationships. In all cases the various strains used in each species represent variants with regard to penicillin production, and all have the same nutritional requirements. All parent strains used grow satisfactorily on a medium containing only salts, inorganic nitrogen, and a carbon source, such as glucose or sucrose. They have no accessory growth factor requirements. The basic, or minimal, medium used is Czapek-Dox medium containing the following ingredients in grams/liter: NaNO3, 3.0; KH2PO4, 1.0; MgSO47H20, 0.5; KCI, 0.5; FeSO4, 0.1; and sucrose, 30.0. To insure growth of new strains having additional nutritional requirements, treated spores were allowed to germinate and grow on a medium supplemented with growth factors. The "complete" medium used is the penicillin sporulation medium of the Northern Regional Researcl 1 Received for publication June 17, 1946. The work reported in this paper was done under the Office of Production Research and Development, War Production Board, Contract 169, and under contract recommended by the Committee on Medical Research, between the Office of Scientific Research and Development and Stanford University. 2 Present address Osborn Botanical Laboratory, Yale University, New Haven, Connecticut. The author wishes to thank Professor G. W. Beadle and Dr. H. K. Mitchell for their many contributions to the work reported. For invaluable assistance in performing the work the author wishes to thank Miss Katherine Ann Bolender, Mrs. Miriam Bonner, Miss Margaret Briggs, Miss Charlotte Brown, Miss Martha Dodds, Miss Ruth Dryer, Miss Marilou Hackley, Miss Dorothy Hackley, Mrs. Mary V. Hungate, Mrs. Nancy Lawrence, Miss Timona Miller, Mrs. Caryl P. Moerdyke, Mr. Adrian Srb, Dr. Lotti Steinitz, and Miss Evelyn Vipham. Laboratory and contains the following ingredielnts in grams/liter: glycerine, 7.5; Brer Rabbit Molasses, 7.5; Bacto Peptone, 5.0; NaCl, L.0; MgSO417H20, 0.05; KH2PO4, 0.06; ferric tartrate, 0.003; CuS04, 0.001; CaCO3, 0.05. This medium is ordinarily solidified with 2 -per cent agar for use as slants, and stock cultures are maintained on it. The vitamin solution contains the following ingredients in milligrams/liter of solution: tliiamin, 100; riboflavin, 50; pyridoxin, 50; pantothenic acid, 200; p-aminobenzoic acid, 50; nicotinamide, 200;