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A M182T substitution was discovered as a second-site suppressor of a missense mutation in TEM-1 ␤-lactamase. The combination of the M182T substitution with other substitutions in the enzyme indicates the M182T substitution is a global suppressor of missense mutations in ␤-lactamase. The M182T substitution also is found in natural variants of TEM-1 ␤-lactamase with altered substrate specificity that have evolved in response to antibiotic therapy. The M182T substitution may have been selected in natural isolates as a suppressor of folding or stability defects resulting from mutations associated with drug resistance. This pathway of protein evolution may occur in other targets of antimicrobial drugs such as the HIV protease.The production of ␤-lactamase is the principal mechanism of bacterial resistance to ␤-lactam antibiotics, such as penicillins and cephalosporins. ␤-lactamase provides resistance by catalyzing the hydrolysis of ␤-lactam antibiotics to ineffective antimicrobials. TEM-1 ␤-lactamase is the most prevalent plasmid-mediated ␤-lactamase in Gram-negative bacteria (1). It is able to efficiently hydrolyze penicillins and most cephalosporins, but not the more recently developed extendedspectrum cephalosporins, such as ceftazidime and cefotaxime (2). In addition, ␤-lactamase inhibitor compounds, such as clavulanic acid, have been developed. These compounds themselves do not possess antimicrobial activity but instead are used in conjunction with other ␤-lactams, such as ampicillin (3). Soon after the introduction of extended-spectrum cephalosporins and inhibitors there were reports of transferable resistance to the drugs (4). Cloning and DNA sequencing revealed that much of the resistance was due to TEM ␤-lactamase enzymes that contained 1-4 amino acid substitutions (4). These substitutions alter the substrate profile of the enzyme such that it can hydrolyze the extended-spectrum cephalosporins or is insensitive to ␤-lactamase inhibitors (4, 5). Interestingly, most of the substitutions found in inhibitorresistant enzymes are different from those found in enzymes able to cleave extended-spectrum cephalosporins (5). An exception appears to be the M182T substitution, which has been identified in both inhibitor-resistant enzymes (TEM-32) and extended-spectrum ␤-lactamases (TEM-43) (refs. 6 and 7; K. Bush, personal communication). In this study, the M182T substitution was identified as a second-site suppressor of an asparagine for leucine substitution at position 76, which is buried in the TEM-1 structure. Further experiments demonstrated the M182T substitution can suppress the effects of deleterious substitutions at other sites in the protein. These findings suggest that the M182T substitution acts as a global suppressor of ␤-lactamase substitutions that disrupt the folding and͞or stability of the enzyme. was used as the host for the assay of antibiotic susceptibility, for immunoblotting, for specific activity measurements, and for the preparation of single-stranded DNA (10). E. coli SB646 [⌬fhuA ⌬ptr ⌬d...

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The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum TEM β-lactamases (ESBLs) with the ability to hydrolyze these drugs. The evolved TEM ESBLs from clinical isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide increased hydrolysis of extended-spectrum cephalosporins or resistance to inhibitors. These active site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability associated with the substitutions. The best studied example is the M182T substitution that corrects protein stability defects and is commonly found in TEM-type ESBLs or inhibitor-resistant variants from clinical isolates. In this study, a genetic selection for second site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clinical isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs that are associated with active site mutations may be a common mechanism for the evolution of novel protein function.