Physical mapping and expression of hybrid plasmids carrying chromosomal beta-lactamase genes of Escherichia coli K-12

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Abstract
Hybrid plasmids carrying the ampC gene of E. coli K-12 that codes for the chromosomal .beta.-lactamase were physically studied. The ampC gene was mapped to a deoxyribonucleic acid segment encompassing 1370 base pairs. The mapping was facilitated by the isolation of a plasmid carrying an insertion of the transposable element gamma delta (.gamma..delta.) close to ampC. The ampA1 mutation, which increases the expression of ampC by a factor of about 20, was localized to a 370-base pair segment of the 1370-base pair DNA segment that contains the ampC gene. Using a minicell protein labeling system, it was seen that plasmids carrying either ampA+, ampC or ampA1 and ampC coded for a 36,000-dalton protein which comigrated with purified chromosomal .beta.-lactamase. In cells carrying plasmids that bore the ampA1 allele, the production of this protein was greater. In additon, a protein with a slightly higher MW (38,000) was expressed by both ampA+ ampC and ampC plasmids in this protein labeling system. This protein might represent a precursor form of chromosomal .beta.-lactamase. From E. coli K-12 strains carrying the ampA1 allele, 2nd-step mutants were isolated that hyperproduced chromosomal .beta.-lactamase. By reciprocal recombination, plasmid derivatives were isolated that carried these mutations. Two 2nd-step regulatory mutations mapped within the same 370-base pair region as ampA1. This piece of DNA therefore contains ampA, a control sequence region for ampC.