Purification of a class C A-type beta-lactamase from a derepressed strain of Enterobacter cloacae. Comparison of the wild-type and mutant enzyme with those from strains P99, 208 and GN7471.

Enterobacter cloacae strain 5822 expresses low levels of a class C beta-lactamase which can be induced 100-fold by imipenem. Mutants that constitutively express high levels of beta-lactamase can be selected on aztreonam or cefotaxime. The beta-lactamase from one such mutant (5822M2) has been purified to homogeneity and compared on the basis of subunit Mr, pI, substrate specificity, inhibitor sensitivity and immunological cross-reactivity with the enzyme from strains P99, GN7471 and 208, which have been studied previously. The enzyme from strain 5822M2 is clearly related to these other forms and is of the A-type according to the criteria of Seeberg, Tolxdorff-Neutzling & Wiedemann [Antimicrob. Agents Chemother. (1983) 23, 918-925]. The enzyme from the wild-type strain (5822) is shown to be identical to that found in the depressed strain (5822M2), indicating that the mutation is in a regulatory gene. A detailed analysis of the kinetics of the enzyme from strain 5822M2 shows that all of the beta-lactams studied are substrates and that a mechanism involving the formation of an acyl-enzyme is probably applicable in every case. The substrates however can clearly be grouped into two classes, i.e. 'good' substrates with kcat. values of 80-1200 s-1 and 'poor' substrates/good inhibitors with kcat. values of 0.009-0.00007 s-1. The permeability barrier to aztreonam is 4-fold less in the derepressed strain when compared with the wild-type strain. This is associated with significant changes in the expression of outer membrane porins. The observed resistance in the derepressed mutant appears to be linked to the elevated levels of beta-lactamase (3000-fold) rather than to the modest changes in the permeability barrier.