Analysis of the effects of -42 and -32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing ampC.
Escherichia coli usually produces only very small amounts of a constitutive AmpC beta-lactamase, but clinical strains overproducing this enzyme have been isolated. Three different ampC promoters of E. coli clinical strains were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene in the pKK232-8 reporter plasmid and their relative strengths were compared by two different methods. The strength of the promoters from AmpC hyperproducers was 70- to 120-fold higher than those from a low-level AmpC producer. One of the strong promoters, which differs from strain K12 at bases -88, -82, -42, -18, -1 and +58, was mutated to abolish the -42 mutation. This change resulted in a 43-fold decrease in CAT concentration. In another promoter, with eight different mutations at positions -88, -82, -32, -18, -1, +5, +24 and +58, the -32T-->A transversion, which created perfect homology with the -35 consensus sequence, was reverted; this led to a 13-fold decrease in CAT concentration. The -42 and -32 mutations play an important role in E. coli resistance to beta-lactams by increasing ampC transcription.
Caroff
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Laboratoire de Bactériologie-Virologie, Faculté de Pharmacie, 1 rue Gaston Veil, 44035 Nantes. Laboratoire de Bactériologie, Virologie, Hygiène hospitalière, CHU, Nantes, France. interepi@chu-nantes.fr
Espaze
E
E
Gautreau
D
D
Richet
H
H
Reynaud
A
A
D001426:Bacterial Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics*
D001426:Bacterial Proteins; D015500:Chloramphenicol O-Acetyltransferase; C048795:AmpC beta-lactamases; D001618:beta-Lactamases