ESCHERICHIA-COLI CHROMOSOMAL MUTATIONS THAT PERMIT DIRECT CLONING OF THE BACTEROIDES-FRAGILIS METALLO-BETA-LACTAMASE GENE, CCRA

The class B, metallo-beta-lactamase genes ccrA (carbapenem- and cephamycin resistance) from three Bacteroides fragilis isolates - QMCN3, QMCN4, and TAL3636 - were cloned and expressed in Escherichia coli. Cloning of the genes, by selecting for ampicillin resistance, was facilitated by two classes of Escherichia coli chromosomal mutations which resulted in at least a 5-10-fold increase in metallo-beta-lactamase enzymatic activity. The observed increase in enzymatic activity is due to either increased translation of the ccrA gene or an effect on localization or stability of the protein. Comparison of the DNA sequences of the three ccrA genes revealed that their protein-coding sequences shared greater than 97% DNA sequence identity. However, the 5' upstream sequence for the TAL3636 ccrA gene was unrelated to that of the other two genes.