Nitrogen regulation of theput operon in enteric bacteria

Enteric bacteria can grow on proline as the sole nitrogen and carbon source. Expression of the proline utilization (put) operon in Klebsiella aerogenes and Escherichia coli is responsive to nitrogen regulation, but Salmonella typhimurium can not activate put operon expression when growing in medium with glucose as a carbon source and proline as the sole nitrogen source.In order to compare nitrogen regulatory sites in the put control regions from these three closely related genera, the put operon from K. aerogenes was cloned and characterized. The overall size and organization of the put control region is very similar in all three bacteria. The same $\sigma\sp{70}$ dependent transcription start sites are used during growth in limiting or excess nitrogen conditions, indicating that the put operon in K. aerogenes is not directly regulated by the ntr system.The put expression from each of these three species was assayed when moved into the other two species. Expression of the put operon indicated that loss of nitrogen regulation in S. typhimurium is due to the absence of both a trans-acting factor (the nac gene product) in the cytoplasm and a cis-acting site in the put regulatory region (the Nac protein binding site). Lack of nitrogen regulation is accentuated in S. typhimurium because catabolite repression is more severe during nitrogen starvation.P22 challenge phage carrying the Nac binding site from the K. aerogenes put regulatory region was used to identify the nucleotide residues important for Nac protein recognition in vivo. Mutations in an asymmetric 30 base pair region prevented DNA binding by Nac protein, and the DNA sequence required for Nac binding is absent from the put regulatory region in S. typhimurium.The Nac protein is a member of the LysR family of DNA binding proteins. To understand how the Nac protein interact with DNA, P22 challenge phage system was used to isolate the second site suppressors of Nac protein that can recognize a specific mutant nac binding site. The second site suppressors of Nac protein substituted an amino acid in the helix-turn-helix region indicating that this DNA-binding motif directly contacts nucleotides in the Nac binding site.U of I OnlyETDs are only available to UIUC Users without author permissio