ADAPTATION TO SULFONAMIDE RESISTANCE IN NEISSERIA-MENINGITIDIS MAY HAVE REQUIRED COMPENSATORY CHANGES TO RETAIN ENZYME FUNCTION - KINETIC-ANALYSIS OF DIHYDROPTEROATE SYNTHASES FROM N-MENINGITIDIS EXPRESSED IN A KNOCKOUT MUTANT OF ESCHERICHIA-COLI

Previously, the effects of three point mutations (at amino acid positi ons 31, 84, and 194) in the gene coding for a sulfonamide-resistant di hydropteroate synthase of Neisseria meningitidis were analyzed by site -directed mutagenesis, Changes at positions 31 and 194 abolished the p henotypic expression of sulfonamide resistance, while a change at posi tion 84 appeared to be neutral. These studies are here extended to cor relate the alterations in phenotype with effects on enzyme kinetics by expressing the cloned meningococcal genes in an Escherichia coli stra in that had its dhps gene partially deleted and replaced by a resistan ce determinant. The most dramatic effects were produced by mutations a t position 31, A change from the Leu found in the resistant isolate to a Phe (the residue found in sensitive isolates) led to a 10-fold decr ease in the K-m and a concomitant drop in the K-i. Changes at position 194 also affected both the K-m and K-i but not to the same extent as mutations at position 31, Changing position 84 altered the K-m only sl ightly but significantly. This latter change was interpreted as a comp ensatory change modulating the function of the enzyme, In another type of resistance gene, 2 amino acid residues, proposed to be an insertio n, were deleted, resulting in a sensitive enzyme. However, the resulti ng K-m was raised 10-fold, suggesting that compensatory changes have a ccumulated in this type of resistance determinant as well. This resist ance gene differs by as much as 10% from the sensitive isolates, which makes identification of important mutations difficult.