Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase

Area of Science:

• Microbial Physiology
• Bacterial Genetics
• Enzyme Regulation

Background:

• Escherichia coli fumarate reductase (frd) and succinate dehydrogenase (sdh) interconvert fumarate and succinate.
• Aerobic induction of fumarate reductase is limited, preventing succinate dehydrogenation.
• The fnr+ gene product activates anaerobic respiratory enzymes.

Purpose of the Study:

• To investigate suppressor mutations enabling aerobic succinate growth in an sdh strain.
• To characterize the regulatory mechanisms of the fumarate reductase (frd) operon.
• To determine the location and nature of mutations affecting frd expression.

Main Methods:

• Selection of three classes of suppressor mutants: frd oxygen-resistant [frd(Oxr)], constitutive [frd(Con)], and gene amplification [frd(Amp)].
• Analysis of fumarate reductase activity under aerobic and anaerobic conditions.
• Genetic analysis using partial diploids and reporter gene fusions (phi(frd+-lac)).

Main Results:

• All selected mutants exhibited increased aerobic fumarate reductase activity.
• frd(Oxr) mutants showed increased resistance to aerobic repression and reduced fnr+ dependence anaerobically.
• frd(Con) mutants displayed high resistance to aerobic repression, fnr+ independence, fumarate independence, and nitrate repression immunity anaerobically.
• Mutations in frd(Oxr) and frd(Con) act in cis, suggesting promoter region involvement.
• frd(Amp) mutants showed gene amplification without significant regulatory changes.

Conclusions:

• Suppressor mutations alter the regulation of the fumarate reductase (frd) operon, enabling aerobic succinate utilization.
• Aerobic and anaerobic nitrate repression mechanisms are distinct.
• cis-acting mutations, likely in the promoter, control frd operon expression.