Certain strains of Bacillus ubtilis and BaciUus mesentericus produce glutamyl polypeptide similar to that produced by Bacilus anthracis (IvAnovics and Erdos, 1937; Bovarnick, 1942). The peptide of the latter organism is contained largely in a capsule and is released into the medium upon autoclaving or upon aging and autolysis of the cells, whereas most, if not all, of that produced by B. subtilis occurs free in the medium. Some of the

Some factors influencing peptide production by Bacillus subtilis were reported in an earlier publication (Thorne et al., 1954). Yields of peptide in excess of 15 mg per ml were obtained in a medium composed of glycerol, citric acid, L-glutamic acid, NH4Cl, MgSO4, FeC13, and K2HPO4 (medium C). Maximum growth and peptide production were obtained only when tap water and a specific lot of FeCl3 were used in preparing the medium. This suggested that unidentified metallic ions in tap water and in the lot of FeCl3 were necessary. The present paper deals with the identification of these ions and presents a chemically defined medium (medium E) for optimum growth and peptide production. It also discusses the effects of various metallic ions on configuration of the glutamic acid comprising the polypeptide synthesized in this medium. MATERIALS AND METHODS Culture and medium. Bacillus subtilis, ATCC 9945A, designated previously by us as strain CDII, was used. The basal medium (designated here as medium C) and methods for producing peptide in shaken flasks were the same as those described previously (Thorne et al., 1954).

Bacilus anthracis produces a capsular polypeptide that is composed of D-glutamic acid (Hanby and Rydon, 1946). During an investigation on factorsaffecting synthesis of this substance, it was observed that large quantities of glutamic acid were synthesized through transamination. The transamination reaction was reviewed by Herbst (1944). The formation of glutamic acid and aspartic acid by the following reactions has been found to occur in a large number of tissues and microorganisms: (1) L-glutamic acid + oxalacetic acid a ± xa-ketoglutaric acid + L-aspartic acid (2) L-glutmatic acid + pyruvic acid a b ketoglutaric acid + L-alanine This paper reports the results of studies on glutamic acid synthesis by intact cells and cell-free extracts of Bacillus anthracis by way of reaction (lb). EXPERIMENTAL METHODS Cultures. The organisms used included Escherichia coli (ATCC 4157), Bacillus subtilis (ATCC 9945), and two strains of Bacillus anthracis, an avirulent mucoid strain, M, and a virulent rough strain, 994. Stock spore suspensions of B. subtilis and B. anthracis were used, and E. coli was maintained on nutrient agar. Test for transaminase activity. Cultures were grown in Roux bottles for 16 to 24 hours at 37 C on nutrient agar. The cells were scraped off the agar, washed twice with saline, and finally suspended in 0.1 M phosphate buffer, pH 7.4. Five ml of washed cell suspension were added to an equal volume of solution containing 1 mM of L-aspartic acid and 1 mm of a-ketoglutaric acid (or other substrates to be tested) at pH 7.4, and incubated at 38 C in an evacuated Thunberg tube for the required time. The contents of the tube were then filtered to remove the cells, and the filtrate was assayed for glutamic acid by the chromatographic method described below. When cell-free extracts were used they were prepared by treating 25 ml of the cell suspension in a Raytheon sonic oscillator for 20 minutes at 9,000 cycles per second. Paper chromatographic method for glutamic acid, aspartic acid, and alanine. The method used by us includes modifications of other published methods (Consden,

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