Regulatory components in Citrobacter freundii ampC beta-lactamase induction

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Citrobacter freundii possesses an inducible chromosomal beta-lactamase, analogous to Escherichia coli's constitutive ampC beta-lactamase.
• In C. freundii, the ampC gene is separated from the fumarate reductase (frd) operon by a DNA segment containing the ampR gene, which encodes a regulatory protein.

Purpose of the Study:

• To investigate the regulatory mechanism of the inducible chromosomal beta-lactamase in Citrobacter freundii.
• To elucidate the role of the AmpR protein in the expression of the C. freundii ampC gene.

Main Methods:

• Cloning of the C. freundii OS60 ampC gene into E. coli.
• Analysis of ampC gene induction by beta-lactam antibiotics in the presence and absence of the ampR gene.
• Isolation and characterization of spontaneous mutants exhibiting constitutive beta-lactamase overproduction.

Main Results:

• The cloned C. freundii ampC gene requires an intact ampR gene for induction by beta-lactams in E. coli.
• The AmpR protein represses C. freundii ampC synthesis 2.5-fold without inducer and enhances expression 11-fold upon beta-lactam addition.
• Spontaneous constitutive ampC overproduction mutants were identified, with mutations located outside the frd-amp region, suggesting additional regulatory factors.

Conclusions:

• The AmpR protein plays a dual role in regulating C. freundii ampC expression, acting as both a repressor and an activator.
• The regulatory system for C. freundii ampC is complex and involves factors beyond the ampR gene.