Isolation and characterization of Escherichia coli birA intragenic suppressors

The biotin (bio) operon in Escherichia coli is negatively regulated by BirA, a bifunctional protein with both repressor and biotin-activating functions. Twenty-five heat-resistant revertants of three temperature-sensitive birA alleles (birA85, birA104 and birA879) were isolated and categorized into five growth and six repression classes. The revertants appear to increase biotin activation by raising the specific activity of BirA and/or increasing the number of enzyme molecules. The 19 birA85 revertants displayed a broad range of activity for both enzyme and repressor functions, and may represent intragenic second-site suppressor mutations. The birA85 revertants included a novel class of bio superrepressor mutations. Repressor titration experiments suggested that many of the birA85 revertants increase BirA concentrations above wild-type levels because the repressors were not competed from the chromosomal bio operator by multicopy bio operator plasmids. The majority of the birA104 revertants resulted in both wild-type repressor and enzyme activity; they are possibly true revertants in which the amino acid residue altered by the birA104 mutation has been substituted by the wild-type or a chemically similar amino acid.