The overall objective of this work has been to understand how the enteric bacteria, especially Klebsiella aerogenes, respond to the quality and quantity of their nitrogen source. The research proposed here is tightly focused on the Nitrogen Assimilation Control Protein (NAC), a LysR-type regulator that activates transcription of ammonia-generating operons like hutUH (histidine utilization) and represses transcription of ammonia-using operons like gdhA (glutamate dehydrogenase). There are 3 specific aims of this proposal. First, a genetic analysis of positive-control (PC) and negative-control (NC) mutants will be carried out. PC mutants will be selected here as those that bind DNA (at the nac promoter) normally but fail to activate hutUp and NC are those that bind but fail to repress at gdhAp. Such mutations will include mutant sites that cannot deform NAC appropriately, mutant NAC that has altered site recognition, and mutant NAC that has altered surfaces required for protein-protein contact. Second, the effect of NAC on the binding and activity of RNAPolymerase will be determined using NAC, PC and NC mutants of NAC, and small (active) fragments of NAC. These will be tested using gel-shift assays (for binding) and run- off transcription assays with normal RNAP and a mutant form with a deletion of the a-CTD. finally the structure of NAC will be probed using chimeras formed between NAC and other LysR proteins (OxyR and CysB). The turnover rate of NAC will be measured and tested to see if it is regulated. And an attempt will be made to confirm whether the N- and C-termini of NAC are in close proximity. If time allows, a system to study oriented heterodimers will also be developed. An attempt to grow crystals of NAC or to purify large quantities of NAC fragments for nmr solution will continue throughout the proposal period. By the end of these 5 years, the data gathered should give hints on 1. how NAC activates transcription and the role of the DNA in causing this activation, 2. how NAC represses transcription (whether there is a NAC-NAC interaction involved and whether the DNA site plays a role in it), and 3. what parts of the NAC polypeptide are involved in communicating among the various interaction sites.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BM-1 (01))
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Anderson, James J
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University of Michigan Ann Arbor
Schools of Arts and Sciences
Ann Arbor
United States
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Bender, Robert A (2010) A NAC for regulating metabolism: the nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae. J Bacteriol 192:4801-11
Frisch, Ryan L; Bender, Robert A (2010) Expanded role for the nitrogen assimilation control protein in the response of Klebsiella pneumoniae to nitrogen stress. J Bacteriol 192:4812-20
Rosario, Christopher J; Frisch, Ryan L; Bender, Robert A (2010) The LysR-type nitrogen assimilation control protein forms complexes with both long and short DNA binding sites in the absence of coeffectors. J Bacteriol 192:4827-33
Frisch, Ryan L; Bender, Robert A (2010) Properties of the NAC (nitrogen assimilation control protein)-binding site within the ureD promoter of Klebsiella pneumoniae. J Bacteriol 192:4821-6
Rosario, Christopher J; Janes, Brian K; Bender, Robert A (2010) Genetic analysis of the nitrogen assimilation control protein from Klebsiella pneumoniae. J Bacteriol 192:4834-46
Goss, Thomas J (2008) The ArgP protein stimulates the Klebsiella pneumoniae gdhA promoter in a lysine-sensitive manner. J Bacteriol 190:4351-9
de la Riva, Lucia; Badia, Josefa; Aguilar, Juan et al. (2008) The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation. J Bacteriol 190:7892-903
Liu, Qiong; Bender, Robert A (2007) Complex regulation of urease formation from the two promoters of the ure operon of Klebsiella pneumoniae. J Bacteriol 189:7593-9
Rosario, Christopher J; Bender, Robert A (2005) Importance of tetramer formation by the nitrogen assimilation control protein for strong repression of glutamate dehydrogenase formation in Klebsiella pneumoniae. J Bacteriol 187:8291-9
Shankar-Sinha, Sunita; Valencia, Gabriel A; Janes, Brian K et al. (2004) The Klebsiella pneumoniae O antigen contributes to bacteremia and lethality during murine pneumonia. Infect Immun 72:1423-30

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