Ten micrograms of 2-nitro-1-butyl pyridinium sulfate (NBPS) per milliliter inhibited the growth of Bacillus subtilis by blocking thymidylate synthetase. The inhibition could be reversed by the addition of thymine or cytosine.2-Nitro-1-butyl pyridinium sulfate (NBPS) is an inhibitor produced by Nalco Chemical Corp. and is used commercially to control crown rot of bananas. We undertook an investigation to determine the mode of action of NBPS, by using Staphylococcus epidermidis and Bacillus subtilis as our test organisms.Growth of S. epidermidis and B. subtilis was inhibited by 10 ,ug of NBPS per ml, but the inhibitory effect could be reversed by dilution to 5 Ag/ml. Respiration of inhibited (100 ,g/ml) and noninhibited cultures was determined in a Warburg respirometer (17). The results ( Fig. 1) indicate that NBPS has relatively little effect on oxygen uptake, and the morphology of the cells did not appear to be affected by NBPS.Saline solution, nutrient broth, and nutrient broth plus 0.03% yeast extract were added to inhibited cultures (10 Mg of NBPS per ml) at different time periods to determine the reversibility of the inhibition. The results (Fig. 2) indicate that the addition of saline solution did not reverse the inhibition. Nutrient broth with 0.03 % yeast extract was more effective in reversing the inhibition than nutrient broth alone.A number of specific products were tested for their ability to reverse the inhibition. The constituents added individually were: 0.03% yeast extract; a basic salts solution (14); hydrolyzed ribonucleic acid, 0.005 and 0.01 %; nicotinamide, 2 and 4 ,g/ml; biotin, 2 and 4 ,ug/ml; p-aminobenzoic acid, 2 and 4 gg/ml; thiamine, 2 and 4 Ag/iml; folic acid, 2 and 4 Mg/ml; pyridoxine, 2 and 6 Ag/ml; nicotinamide adenine dinucleotide, 2 and 4 ,ug/ml; thymine, 25, 50, 100, 150, 200, 250, and 300 ,g/ml; and tryptophan, 50, 100, and 200 MAg/ml. The four I Present address: Botany, Provo, Utah 84601. remaining bases, adenine, guanine, cytosine, and uracil, were added separately in amounts of 25, 50, 100, 200, 250, 300, and 400 Ag/lml. Cytosine and thymine were added together to inhibited cultures in concentrations of 10, 20, 30, 40, 50, 60, and 70 Ag/ml to check for a synergistic reversing action of these two bases. Of the various factors tested for reversibility, only thymine and cytosine reversed the inhibition. When thymine and cytosine were assayed together, no synergistic effect was observed. Thymine, at 50 ,g/ml, effectively reversed the inhibitory action of 10 Mg of NBPS per ml.Thymine-2-'4C was fed to a thymineless mutant of B. subtilis growing in Spizizen's medium (16) containing various concentrations of NBPS (0 to 30 ,g/ml). The results shown in Fig. 3 indicate that NBPS markedly retarded the uptake of thymine into deoxyribonucleic acid (DNA). Thymidine kinase and thymidylate synthetase were isolated and purified by streptomycin sulfate precipitation, differential centrifugation, diethylaminoethyl cellulose, and Sephadex chromatography. The assay mixture for thymidine kinase...