The proposed research is to investigate the structure and regulation of the genes coding for the biodegradative amino acid decarboxylases. These amino acid decarboxylases are produced in large quantity (up to several percent of cell protein) under growth at low pH in rich media. Mu-lac fusions to the arginine and lysine decarboxylase genes have been prepared and will be used to study the transcriptional regulation of these genes. One aspect of these studies will examine expression under various culture conditions, and with use of known global regulatory gene mutants to seek potential physiological effectors of the expression. To identify genes involved in the regulatory response, transposon mutagenesis will be used. Mutants of the lac fusion strain will be sought which exhibit altered expression in response to air or pH. The positions of these mutations can be mapped and the regulatory protein identified through complementation and cloning experiments. Overproduction of regulatory proteins (e.g., cadR, a regulator of lysine decarboxylase) using high expression plasmid based systems is planned. Structural studies of the cadA gene (lysine decarboxylase) and other genes will yield information on the sequence of the regulatory region and the evolutionary similarity of this protein to other decarboxylases. In vitro mutagenesis and studies using defined expression systems will allow the DNA sites required for pH, air and amino acid effects to be located. Eventually, in vitro studies of the interaction of the transcription regulatory proteins with the DNA will further define the position and steps involved in the regulatory response. Comparison of sequences and regulatory factors between these similarily pH responsive genes should allow the general features of this novel type of regulation to be revealed.
