The araIc mutation in Escherichia coli B/r.

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• The araBAD promoter in Escherichia coli is regulated by the araC activator protein.
• Previous studies identified araIc mutants as constitutive expressors of araBAD genes.
• These mutants were initially isolated as Ara+ "revertants" from an araC deletion mutant.

Purpose of the Study:

• To characterize the molecular basis of the araIc mutation.
• To define the cis-acting elements responsible for constitutive araBAD gene expression.

Main Methods:

• Isolation of nineteen araIc mutants.
• Cloning of eight araIc mutations onto pBR322-ara hybrid plasmids using in vivo recombination.
• Restriction enzyme analysis and DNA sequencing of the cloned mutations.

Main Results:

• Eight araIc mutations were successfully cloned.
• Restriction and DNA sequence analysis revealed a single base-pair change in each of the analyzed mutations.
• This critical base-pair alteration is located at the -35 region of the araBAD promoter.

Conclusions:

• The araIc allele represents a cis-acting mutation within the araBAD promoter.
• A single base-pair change at the -35 promoter region is sufficient to cause constitutive expression of araBAD genes.
• This finding provides a precise molecular definition of the araBAD promoter's regulatory elements.