Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after Nmethyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms.

The degree of participation (DP) of exogenous thymidine and thymine in overall DNA synthesis was studied in Lactobacillus acidophilus R-26. The DP of thymidine remains constant under a variety of conditions (except at low thymidine concentrations, when the DP is influenced by deoxyribonucleosides and folic acid). A 5-bromodeoxyuridine-resistant mutant was isolated, which displayed cross-resistance to 5-bromouracil and a significantly lower DP of thymidine than the parental strain. Thymine was poorly incorporated in the parental strain even in the presence of deoxyribosides. The results of this investigation would be compatible with the possibility of an alternative pathway for thymidylate synthesis other than the known thymidylate synthase pathway.

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