RESISTANCE TO OXYIMINO BETA-LACTAMS DUE TO A MUTATION OF CHROMOSOMAL BETA-LACTAMASE IN CITROBACTER-FREUNDII

The duplicative mutation of an Ala-Val-Arg sequence at positions 208 t o 210 in the loop structure of Enterobacter cloacae class C beta-lacta mase caused substrate specificity extension to oxyimino beta-lactam an tibiotics and this chromosomal mutation provided bacterial cells with high resistance to the beta-lactams (M. Nukaga et al, 1995, J. Biol. C hem, 270, 5729-5735). In order to confirm the universality of this phe nomenon among other class C beta-lactamases, the duplicative mutation was applied to a class C beta-lactamase of Citrobacter freundii, which has 74% homology to the E. cloacae beta-lactamase amino acid sequence . The counterpart sequence to the Ala-Val-Arg of the E. cloacae enzyme in C. freundii beta-lactamase was identified to be Pro-Val-His. A Pro -Val-His sequence was inserted just after the native Pro-Val-His seque nce at positions 208 to 210 in the C. freundii beta-lactamase. The res ulting mutant of C. freundii beta-lactamase obtained a striking charac teristic that we expected, showing substrate specificity extension to oxyimino beta-lactams. Nearly the same result was obtained with the in sertion of an Ala-Val-Arg sequence after the native Pro-Val-His sequen ce. These results indicate that structural modification of this locus commonly induces modification of the substrate specificity to unfavora ble substrates for many chromosomal class C beta-lactamases produced b y Gram-negative bacteria.