Signalling proteins in enterobacterial AmpC beta-lactamase regulation

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The ampC beta-lactamase from Citrobacter freundii is inducible in Escherichia coli, regulated by the ampR gene.
• Basal expression and inducibility of ampC beta-lactamase are influenced by the E. coli proteins AmpD and AmpE.

Purpose of the Study:

• To investigate the roles of the ampD and ampE genes in regulating ampC beta-lactamase expression and beta-lactam resistance in E. coli.
• To elucidate the mechanism by which AmpD and AmpE proteins interact with the regulatory pathway involving AmpR.

Main Methods:

• Construction and analysis of E. coli deletion mutants for ampD and ampE genes.
• Introduction of specific mutations, including an out-of-frame deletion in AmpD and an IS1 insertion in ampD.
• Expression of AmpE from a recombinant plasmid in an ampD-ampE deletion mutant.
• Assessment of beta-lactamase expression levels and beta-lactam resistance.

Main Results:

• Deletion of both ampD and ampE resulted in constitutive, ampR-dependent overproduction of beta-lactamase.
• Mutations in ampD led to increased basal beta-lactamase expression and altered inducibility.
• AmpE expression in an ampD-ampE deletion mutant reduced basal beta-lactamase expression but increased hyperinducibility, suggesting AmpD modulates AmpE's effect.
• AmpD and AmpE are not beta-lactam-binding proteins but likely relay signals related to peptidoglycan biosynthesis to AmpR.

Conclusions:

• AmpD and AmpE play crucial roles in modulating the basal expression and inducibility of ampC beta-lactamase.
• These proteins do not directly bind beta-lactams but likely function as signal transducers involved in sensing cell wall synthesis.
• The interplay between AmpD and AmpE influences beta-lactam resistance by affecting the regulatory response of beta-lactamase production.