Analysis of the Effects of -42 and -32 ampC Promoter Mutations in Clinical Isolates of Escherichia Coli Hyperproducing ampC

J Antimicrob Chemother. 2000 Jun;45(6):783-8. doi: 10.1093/jac/45.6.783.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins*
  • Chloramphenicol O-Acetyltransferase / genetics
  • Escherichia coli / genetics*
  • Microbial Sensitivity Tests
  • Mutagenesis, Site-Directed / genetics
  • Mutation / genetics*
  • Plasmids / genetics
  • Promoter Regions, Genetic / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction
  • beta-Lactamases / biosynthesis*
  • beta-Lactamases / genetics*

Substances

  • Bacterial Proteins
  • Chloramphenicol O-Acetyltransferase
  • AmpC beta-lactamases
  • beta-Lactamases