Among spontaneous mutants of Escherichia coli selected for resistance against sulfonamides, thermosensitive strains were found. These were shown to possess a changed dihydropteroate synthase (EC 2.5.1.15), which had a substantially higher Km value for its normal substrate, p-aminobenzoic acid, and an about 150fold higher Km for sulfonamides. The mutationally changed dihydropteroate synthase was found to be thermosensitive by in vitro assays. The thermosensitivity was used as an enzyme marker to demonstrate the complex formation between 2-amino-4-hydroxy-6-pyrophosphorylmethyl pteridine and sulfonamides by partially purified dihydropteroate synthase. The formation of folate from 2-amino-4hydroxy-6-pyrophosphorylmethyl pteridine and p-aminobenzoylglutamic acid by dihydropteroate synthase was found to be very sensitive to inhibition by sulfonamides and very inefficient with the mutationally changed enzyme.The formation of dihydropteroate from paminobenzoic acid (AB) and reduced 2-amino-4hydroxy-6-hydroxymethyl pteridine (pteridine-CH20H) has for a long time been known to be the target of sulfonamide action (4). Part of the inhibitory effect of sulfonamides on bacterial growth has been ascribed to the incorporation of the drug into a pteridine complex to give a sulfonamide-containing H2-pteroate (7,8-dihydropteroic acid) analog (3,4). These investigations, however, have not linked the analog formation to the activity of dihydropteroate synthase (EC 2.5.1.15).In a previous study on plasmid-mediated sulfonamide resistance, the occurrence of a plasmid-borne, drug-resistant dihydropteroate synthase was described (15). Spontaneous mutants of Escherichia coli were isolated that carried temperature sensitivity lesions. These were allegedly located in the chromosomal dihydropteroate synthase gene and were used as tools for the identification of extrachromosomal synthase activity.Experiments are described here which demonstrate the temperature sensitivity of isolated mutant dihydropteroate synthase activities. Partially purified enzyme from these mutants catalyzed the formation of a sulfonamide-containing complex from 2-amino-4-hydroxy-6-pyro-phosphoryl-methyl pteridine (H2-pteridine-CH20-PP) and [35S]sulfathiazole, as did the wild-type enzyme, but the formation of sulfonamide com-plex was temperature sensitive with mutant enzyme.The temperature-sensitive mutants were originally isolated as sulfonamide-resistant strains of E. coli C (15). The temperature sensitivity was interpreted to be a consequence of the mutational change in the enzyme to sulfonamide resistance. In concurrence with this interpretation, dihydropteroate synthase isolated from the mutants showed a Km value about 150-fold higher for sulfathiazole than that isolated from the wild type.The efficiency of the enzymatic formation of dihydrofolate from H2-pteridine-CH20-PP and N-(p-aminobenzoyl)-L-[ U-14C]glutamate (ABG) was also seen to decrease drastically as a consequence of mutational change in the dihydropteroate synthase. MATERIALS AND METHODSBacteria. The E....

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