The crude solid residue was recrystallized from methanol ether and gave 49 g. (56%) of product, m.p. 234-237'.Anal. Calcd. for CCH15C1S2O: C, 43.5; H, 9.0; C1, 21.3; N, 16.8. Found: C, 43.1; H, 9.0; C1, 21.0; S, 16.4. 0-Bromoamides .-The amides listed in Table I were made by reaction of acid chlorides with ammonia or amines either in aqueous or in anhydrous media. If a concentrated aqueous solution of the base was available commercially, the aqueous procedure was used. Ether or chloroform generally was used for the anhydrous bases. After filtration of the hydrochlorides of the bases the products were obtained by evaporation of the solvent and purifiea in the manner indicated in Table I. 2-Ethyl-2-( 2 -bromo-2-ethy1butyramido)-butyrarnide (LV) .-To a n ice-cold, stirred solution of 35 g. (0.21 mole) of 2-amino-2-ethylbutyramide hydrochloride in 300 cc. of Ivater and 50 cc. of acetone there was added, simultaneously, during 1 hour, a solution of 47.1 g. (0.22 mole) of 2-bromo-2-ethylbutyryl chloride in 100 cc. of acetone and 490 cc. (0.49 mole) of 1 N sodium hydroxide solution. The product separated as a white solid which was filtered and airdried. N-(2-Bromo-2-ethylbutyryl)-glycine (LII) .-To a solution of 2.8 g. (0.01 mole) of N-(2-bromo-2-ethylbutyryl)-glycine ethyl ester23 in 25 cc. of methanol, there was added, dropwise, a t 5", with stirring, 2 cc. (0.01 mole) of 5 N sodium hydroxide solution. After having been stored a t room temperature overnight the solution was concentrated under reduced pressure to remove most of the methanol and the (23) K. W. Rosenmund, Ber., 43, 4470 (1909).rryulting solutio11 was acidified with dilute l~ydrochloric acid to pH 1. The product separated from the coaled solution as a white precipitate which was filtered, n a 4 i e d with water, and dried. 1-(2-Bromo-2-ethylbutyryl)-3-methylurea (LVII I .-Amixture of 25 g. (0.34 mole) of N-methylurea and 36.1 g. (0.12 mole) of 2-bromo-2-ethylbutyryl chloride was heated on a steam-bath for six hours with occasional swirling. A clear reddish-brown solution formed to which 1% as added 100 g. of ice and 50 cc. of water. A gum formed nhich solidified upon trituration. The solid was filtered and washed first with cold water and then with petroleum ether (b.p. 20-40"). The crude solid was extracted with petroleum ether in a Saxhlet apparatus for 4 hours. Concentration of the solution t o a small volume gave the product as a white solid. 1-(2-Bromo-2-methylpropionyl)-3-methylurea (XXIII).-A mixture of 20 g. (0.12 mole) of 2-bromo-2-methylpropionyl chloride and 17.8 g. (0.24 mole) of methylurea was heated on a steam-bath for 0.5 hour. An oily solid formed and the mass was treated with water and -filtered. T h e white solid was dried on a porous plate.l-(2-Bromo-2-methylbutyryl)-3-methylurea (XXX).--A mixture of 37.1 g. (0.5 mole) of dried methylurea and 39.9 g. (0.2 mole) of 2-bromo-2-methylbutyryl chloride u as stirred and heated for 2 hours in a n oil-bath a t 60-87".To the mixture, consisting of a n amher liquid containing some colorless c...

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a, e-Diaminopimelic acid was first isolated from acid hydrolyzates of Corynebacterium diphtheriae by Work (1951). Subsequent studies by Dewey and Work (1952) and by Davis (1952) have demonstrated that a, e-diaminopimelic acid (DAP) can be decarboxylated to lysine and that the path of lysine synthesis apparently involves DAP as an intermediate. Davis (1952) reported that a mutant strain of Escherichia coli (173-25), isolated by the penicillin method (Lederberg and Zinder, 1948; Davis, 1948), had an absolute requirement for DAP and a relative requirement for ilysine. Growth in the absence of lysine was slow and there appeared to be a minimum requirement of lysine for rapid growth in the presence of DAP. The work reported here is an investigation of the role of diaminopimelic acid in the nutrition of the DAP auxotroph E. coli strain 173-25. MATERIALS AND METHODS E. coli strain 173-25 (Obtained from Dr. B. Davis, New York University) was maintained at 4 C after monthly transfer on a stock agar containing: K2HHPO4, 0.3 g; yeast extract, 0.1 g; vitamin-free casein hydrolyzate, 20 ml; agar, 1.5 g; and water, 80 ml. Glucose and DAP were added aseptically to a final concentration of 8 mg/ml and 20 ,g/ml, respectively. All growth experiments were conducted in a basal medium consisting of K2HPO4, 7.0 g; KH2PO4, 3.0 g; (NH4)2SO4, 1.0 g; MgSO4-7H20, 0.1 g; lysine, 20 mg; water to 1 L. Five ml of the 2 x concentrated basal medium was dispensed in 18 by 195 mm tubes. Glucose and lysine were added to a final concentration of 8 mg/ml and 20 ,g/ml. The tubes were diluted to a total vol-' While this manuscript was in preparation Dr.

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Tryptophan has long been known as an essential amino acid for many forms of life. However, nuimerous microorganisms do not require nitrogen in the form of tryptophan, and several nitrogenous compounds are known which replace the nutritional requirement for tryptophan. The demonstration that anthranilic acid (Snell, 1943) or indole (Schweigert et al., 1946) supports growth of Lactobacillus arabinosus in the absence of tryptophan suggests the existence of an anthranilic acid-* indole-÷ tryptophan pathway of synthesis. Such a metabolic pathway has been described in Neurospora (Tatum and Bonner, 1943; Tatum, Bonner, and Beadle, 1943), and cell-free extracts of Neurospora sitophila form tryptophan in the presence of indole, serine, and pyridoxal phosphate (Umbreit, Wood, and Gunsalus, 1946). Since serine stimulates the growth of L. arabinoms in the presence of indole (Schweigert, 1947), it has been concluded (Rydon, 1948; Work and Work, 1948) that, as in Neurospora, indole is condensed with serine to yield tryptophan by this organism and, moreover, that anthranilic acid is probably the precursor of indole. Thus, essentially by analogy, the apparent pathway of tryptophan synthesis in Neurospora has been claimed to exist in bacteria. This study was initiated to clarify the biochemical steps leading from anthranilic acid to tryptophan in L. arabinosus. The growth supporting capacity of a series of potential intermediates along the pathway of anthranilic acid bioconversion was tested, and conditions for the utilization of anthranilic acid and indole were studied. The tryptophan content of cells grown in the presence of anthranilic acid, indole, or tryptophan was also determined. The results appear to cast doubt upon the simple metabolic pathway of anthranilic acid conversion to tryptophan previously described. MATERIALS AND METHODS Lactobacillus arabinosus, strain 17-5, from the Cornell University Agricultural Experimental Station, and Leuconostoc mesenteroides, strain P-60, from Merck and Company (through the courtesy of Dr. J. L. Stokes), were maintained as stab cultures at 4 C in the following stock agar: tryptone, 10 g; yeast extract, 10 g; K2HPO4, 5 g; CaCO3, 3 g; glucose, 1 g; liver extract, 20 per cent (by volume); distilled water, 800 ml. The cultures were passed semimonthly for 12 to 15 hours in medium A consisting of tryptone, 10 g; yeast extract, 10 g; glucose, 10 g; K2HPO4, 5 g; distilled water, 1 liter; 20 ml salts solution (see hereafter) and then returned to stock agar. All cultures were incubated in a 37 C water bath.

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