SINGLE AMINO-ACID REPLACEMENTS AT POSITIONS ALTERED IN NATURALLY-OCCURRING EXTENDED-SPECTRUM TEM BETA-LACTAMASES

By directed mutagenesis, we constructed a set of seven TEM-1 derivativ es containing single replacements in each one of the amino acids subst ituted in naturally occurring extended-spectrum TEM beta-lactamases. T he exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities wer e studied. Five of seven mutations determined to some extent variation s in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leak y variation in activity towards cephalosporins and aztreonam. Replacem ents at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The i n vitro construction of mutations appearing in naturally occurring TEM -beta-lactamases, studied in the same genetic context, may help to und erstand the evolution of extended-spectrum beta-lactamases.