Studies on thecad operon of Escherichia coli K-12: ApH regulatory system in bacteria
Doctor of Philosophy
The expression of biodegradative lysine decarboxylase of E. coli is induced in cells grown at low pH, and the maximum induction was observed in the presence of excess lysine under anaerobic conditions. The cadA gene encoding the lysine decarboxylase resides in the cadBA operon. The sequence of about 5 kb of the cadBA operon (3.7 kb) and its flanking regions has been determined. The cadB gene encodes a protein very similar to the ArcD protein, the arginine: ornithine antiporter of Pseudomonas aeruginosa. Both cadA and cadB genes have been subcloned into different expression vectors and the protein products have been expressed and observed in maxicell experiments. The cadB gene has been found to facilitate the excretion of cadaverine, one of the end products of the enzymatic reaction of CadA (lysine decarboxylase), and therefore CadB probably encodes a lysine: cadaverine antiporter. A model for detoxification of extracellular high H$\sp+$ concentration based on the functions of CadA and CadB is proposed. Studies on regulation of the cad operon were performed using lac fusion techniques. A high affinity pH-responsive site(s) was found to be located over 100 bp upstream from the cad promoter by a series of deletion and mutation experiments, and the involvement of a positive pH regulator was confirmed in protein titration experiments. Random mutagenesis and in vivo footprinting were conducted to determine the importance of specific residues in the cad upstream region. The residues detected in these studies of the cad promoter region suggest a complex regulatory mechanism. The possible routes of regulation by the availability of lysine and oxygen were also explored. Hypothetical models account for experimental results are presented.