Lesions at the birA locus of Escherichia coli produce, in varying degrees, derepression of the biotin operon and an increased minimum biotin growth requirement (Barker & Campbell, 1980) as well as diminished biotin uptake and defective biotin holoenzyme synthetase activity (Campbell et al., 1972, 1980). In the accompanying paper, we showed that three birA mutants produce biotin holoenzyme synthetase with altered in vitro properties and that they carry lesions in the structural gene for this enzyme. The pleiotropic birA defect was attributed to structural interactions between a protein domain which includes the holoenzyme synthetase active site and a second protein domain, possibly part of the same polypeptide, which functions as the bio repressor.

To determine if one or more genes reside at birA, we tested pairwise combinations of nine mutations with representative phenotypes for their ability to establish repression of bio expression. The mutations define a single complementation group. Instances of partial complementation appear to be intracistronic, suggesting that the birA product forms a multimer active as both biotin holoenzyme synthetase and repressor.

DNA segments that include and express the birA gene have been cloned into multicopy plasmids. Plasmid-mediated expression of birA can produce a state of superrepression of the bio operon and a concomitant increase in holoenzyme synthetase specific activity. The complementation properties of derivative plasmids, with insertions of Tn5 or small deletions in the bacterial DNA segment, define a 1.6 × 10³ base region that includes the birA gene and a 0.9 × 10³ base segment essential to biotin holoenzyme synthetase and repressor function. The region is flanked by the thrT and tufB genes in a previously unassigned region of the bacterial DNA carried by λdrif^d18.

A preparation of holoenzyme synthetase, purified nearly 10,000-fold, contains a protein that binds specifically to biotin operator DNA as determined by its ability to protect a TaqI endonuclease site that borders the imperfect inverted repeat where the bio repressor is presumed to bind. Biotinyl 5′-adenylate or biotin plus ATP are more effective corepressors than biotin alone, suggesting that biotinyl 5′-adenylate, a presumed intermediate in the holoenzyme synthetase reaction, is the true corepressor.