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Amino acid substitutions in a variant of IMP-1 metallo-beta-lactamase

S Iyobe1, H Kusadokoro, J Ozaki

  • 1Laboratory of Drug Resistance in Bacteria, Gunma University School of Medicine, Maebashi, Japan. siyobe@akagi.sb.gunma-u.ac.jp

Antimicrobial Agents and Chemotherapy
|July 18, 2000
PubMed

Insights

Researchers discovered a new metallo-beta-lactamase variant, IMP-3, with reduced carbapenem-hydrolyzing activity. This finding suggests bla(IMP-3) is an ancestor of bla(IMP-1) due to a single-base mutation.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Enzymology

Background:

  • Metallo-beta-lactamases (MBLs) are enzymes conferring antibiotic resistance.
  • The carbapenem-hydrolyzing beta-lactamase gene bla(IMP) is a significant concern in pathogenic bacteria.
  • Previous studies have identified various IMP variants with differing hydrolytic activities.

Purpose of the Study:

  • To identify and characterize novel variants of the carbapenem-hydrolyzing metallo-beta-lactamase gene bla(IMP).
  • To investigate the genetic basis and functional implications of observed differences in hydrolytic activity between IMP variants.
  • To determine the evolutionary relationship between newly identified IMP variants and known ones.

Main Methods:

  • Polymerase chain reaction (PCR) was used to survey for bla(IMP) genes in pathogenic bacteria.
  • Direct sequencing of PCR products was performed to analyze the bla(IMP-3) gene sequence.
  • Recombinant DNA techniques were employed to construct hybrid bla genes for comparative analysis.
  • Kinetic parameters of beta-lactamases were determined under controlled experimental conditions.

Main Results:

  • A novel metallo-beta-lactamase variant, IMP-3, was detected, exhibiting reduced hydrolysis of penicillins and carbapenems compared to IMP-1.
  • The bla(IMP-3) gene was found on a cassette within a class I integron on the pMS390 plasmid.
  • Nucleotide sequencing revealed seven base substitutions between bla(IMP-3) and bla(IMP-1), with two causing amino acid alterations.
  • A single amino acid substitution (glycine for serine at residue 196) in IMP-3 was identified as responsible for its inefficient hydrolysis of specific beta-lactams.
  • This amino acid change correlated with a single-base mutation (G to A at position 640) in the bla(IMP-3) gene.

Conclusions:

  • The bla(IMP-3) gene, with its reduced hydrolytic activity, is likely an ancestral form of bla(IMP-1).
  • A single-base mutation leading to an amino acid substitution is sufficient to alter the substrate profile of IMP metallo-beta-lactamases.
  • Understanding these evolutionary mechanisms is crucial for tracking the spread of antibiotic resistance genes.

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