This proposal describes experiments aimed at understanding the regulation of expression of the Bacillus subtilis glnA gene. This gene codes for glutamine synthetase (GS), a key enzyme in nitrogen metabolism and one that may play some role in triggering the organism to differentiate (sporulate) in the presence of nitrogen limiting conditions. The glnA gene was the first B. subtilis gene to be characterized with respect to the nature of its regulation by nitrogen and many questions regarding its control remain unanswered. Early work had described GS as playing a direct role in its own control, but other factors have been implicated. We will rigorously characterize the regions of glnA DNA necessary for nutritional control by genetic and biochemical approaches using methylation protection and deletion mapping analyses and alter these sequences by site-directed mutagenesis. The 490 base-pair region of DNA located between the glnA promoter and the start of the GS coding sequences will also be analyzed to determine cis or trans acting roles. Continuous culture experiments will allow us to measure energy charge, GS and glnA message under varied growth conditions enabling us to determine whether ATP-dependent transcription termination near the glnA ribosome binding site is physiologically significant. Once we have established conditions for transcription inhibition in vitro, we will identify, isolate and characterize regulatory components in order to determine how these components act to regulate transcription and what role GS plays in control. Finally, we will determine the chromosomal location of regulatory factor genes that may be unlinked to glnA. The proposed research will answer key questions regarding glnA control and we have will taken a large step towards understanding the mechanisms involved in the regulation of gene expression in a gram-positive, differentiating organism.
