Escherichia coli K-12 mutants hyperproducing chromosomal beta-lactamase by gene repetitions

Abstract

E. coli K-12 ampicillin-resistant mutants hyperproducing chromosomal β-lactamase arose spontaneously from strains carrying ampA1 ampC+. Such mutants were found even in a recA background. Two Amp(r)-100 strains were analyzed genetically. The Amp(r)-100 resistance level of both strains could be transduced by direct selection for ampicillin resistance. Several classes of ampicillin-resistant transductants were found that differed from one another in the β-lactamase activity and the ampicillin resistance mediated by an ampA1 ampC+-carrying strain. The data suggested that β-lactamase hyperproduction was due to repetitions of the chromosomal amp genes. The size of the repeated region was calculated from cotransduction estimates, using the formula of Wu, and was found to be about 1 min in one strain and 1.5 min in the other. Second-step Amp'-400 mutants were isolated from an Amp(r)- 100 strain. The resistance of these mutants was apparently also due to repetitions, each mediating a resistance to about 10 μg/ml. Mutants of wild-type strains that were moderately resistant to ampicillin also gave rise to intermediate-resistance classes, suggesting repetitions of the wild-type amp alleles. F' factors hyperproducing chromosomal β-lactamase by gene repetitions were constructed. They mediated levels of ampicillin resistance comparable to that of naturally occurring resistance plasmids. The expression of β-lactamase hyperproduction was not affected by the presence of ampA and ampC alleles in trans and did not act in trans on the other alleles.