X-Ray Induced Growth Factor Requirements in Bacteria

The specific growth factor requirements of bacteria result from the in- ability of these organisms to synthesize particular essential substances from simpler constituents.1' 2 In some instances such deficiencies have been shown to result from an inability to carry out definite chemical reactions in a biosynthesis.3These defects in synthesis are characteristic of each cell of a given strain of bacteria, and are in addition transmitted to each descendant cell.Similar defects in certain biosyntheses have been in- duced in the ascomycete Neurospora by irradiation and have been shown to result from mutations in specific genes.4 5This suggests that if bio- chemical reactions in bacteria are likewise under genetic control, mutations in certain "genes" in these organisms should lead to corresponding growth factor deficiencies in the modified strains.Treatment with x-rays might be expected to induce such "gene" mutations in bacteria.This paper presents evidence that x-ray treatment of bacteria does produce mutant types, presumably genic, with altered growth factor requirements.6Two species of bacteria have been used in this investigation: Escherichia coli (K-12) and Acetobacter melanogenum (M.A. 6.1).The minimal medium used for E. coli contained per liter: NH4CL, 5.0 g.; NH4NO3, 1.0 g.; Na2S04, 2.0 g.; MgS047H20, 0.1 g.; CaC12, trace; K2HP04, 3.0 g.; KH2PO4, 1.0 g.; 1 cc.trace element solution7; glucose, 5.0 g.; and vitamin- free l-asparagine, 1.5 g. (optional). A. melanogenum has been found to grow well on a minimal medium adjusted to pH 6 with acetic acid and containing per liter: K2HP04, 0.5 g.; KH2PO4, 0.5 g.; MgS04 7H20, 0.2 g.; FeSO4, MnSO4 and NaCl, 0.01 g. each; (NH4)2S04, 10 g.; glycerol, 50 g.; thiamin chloride hydrochloride, 1 mg.; pantoyl l-lactone, 1 mg.; nicotinic acid, 1 mg.; p-aminobenzoic acid, 0.1 mg.; and l-proline, 0.2 g. (optional).The bacteria were treated with x-rays produced in a Westinghouse "Duocondex" unit containing an industrial type oil-cooled tube operated at 160 kv. and 25 ma.Twenty-four or 48-hour-old liquid cultures of bac- teria were exposed to a total irradiation of 180,000 r units given at a rate of 4000 per minute.Preliminary tests had shown that at this dosage over 99.99 per cent of the cells were killed.The irradiated suspensions were then incubated for four hours in yeast extract broth on a shaker.This procedure was adopted to increase the probability of any individual cells altered by the treatment giving rise to homogeneous colonies.Samples of these suspensions were plated out in complete agar medium containing AAA