ampR gene mutations that greatly increase class C β-lactamase activity in Enterobacter cloacae

The ampC and ampR genes of Enterobacter cloacae GN7471 were cloned into pMW218 to yield pKU403, Four mutant plasmids derived from pKU403 (pKU404, pKU405, pKU406, and pKU407) were isolated in an AmpD mutant of Escherichia call ML4953 by selection with ceftazidime or aztreonam. The beta-lactamase activities expressed by pKU404, pKU405, pKU406, and pKU407 were about 450, 75, 160, and 160 times higher, respectively, than that expressed by the original plasmid, pKU403. These mutant plasmids all carried point mutations in the ampR gene. In pKU404 and pKU405, Asp-135 was changed to Asn and Val, respectively. In both pKU406 and pKU407, Arg-86 was changed to Cys, The ease of selection of AmpR mutations at a frequency of about 10(-6) in this study strongly suggests that derepressed strains, such as AmpD or AmpR mutants, could frequently emerge in the clinical setting.