We have a longstanding interest in microbial nitrogen metabolism and its regulation and have focused on the transcriptional activator NtrC (nitrogen regulatory protein C) in enteric bacteria. Products of two of the most highly expressed operons under NtrC control, the glnK amtB and rut operons, are the focus of this proposal. The AmtB (ammonium transporter B) protein and Amt proteins generally appear to be biological gas channels for NH3 rather than active transporters for the ion NH4+, as was previously believed. The AmtB protein is essential for rapid growth of enteric bacteria when the external concentration of NH3 is limiting (>or equal to 50 nM) and is functionally coupled to the high-affinity NH3 assimilatory enzyme glutamine synthetase (GS). One long-term goal of this project is to understand how the E. coli AmtB protein can improve on unmediated diffusion of NH3 across its cytoplasmic membrane. We will use genetic and biochemical approaches to test the hypothesis that AmtB and GS must be in physical contact for AmtB to function, even though their binding must be labile. We will first isolate mutations that disrupt contact between AmtB and GS-called amtB* and glnA*, respectively-and then isolate suppressor mutations that restore contact by altering the partner protein in a compensatory manner. We will initiate studies of the two Amt proteins of the photosynthetic proteobacterium Rhodospirillum rubrum to determine whether one is coupled to GS and the second to biosynthetic glutamate dehydrogenase, the other major NH3 assimilatory enzyme. In a broader context, Amt proteins (called Mep in some organisms) are found widely in microbes, vascular plants, and invertebrate animals and are the first biological gas channels to be described. They are the only specific transporters known for """"""""ammonium"""""""" (used to designate NH3 + NH4+), which is a preferred nitrogen source for many microbes. The association of Amt/Mep proteins with other proteins bears on how gas channels can improve on unmediated diffusion of gases through membranes and on the role of labile supramolecular structures in cell physiology. Our second long-term goal is to continue studies of the b1012 operon. This operon of 7 genes encodes a previously undescribed pathway for degradation of pyrimidine rings, which we have designated the rut (pyrimidine utilization) operon. The adjacent gene, b1013, encodes a negative auxiliary regulator of rut transcription, now called RutR. We will use a variety of genetic and biochemical approaches to identify the intermediates of the Rut pathway and the enzymatic activities of the RutA-RutF proteins. We will study the DNA binding and transcriptional repression activities of RutR in vitro. New biochemical pathways are rare and their characterization is useful in the annotation of genomes and in studies of the metabolic and regulatory interlocks important to cellular self-replication.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Anderson, James J
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University of California San Diego
Schools of Arts and Sciences
La Jolla
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Hall, Jason A; Yan, Dalai (2013) The molecular basis of K+ exclusion by the Escherichia coli ammonium channel AmtB. J Biol Chem 288:14080-6
Vo, Jason; Inwood, William; Hayes, John M et al. (2013) Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12. Proc Natl Acad Sci U S A 110:8696-701
Kim, Minsu; Zhang, Zhongge; Okano, Hiroyuki et al. (2012) Need-based activation of ammonium uptake in Escherichia coli. Mol Syst Biol 8:616
Hall, Jason A; Kustu, Sydney (2011) The pivotal twin histidines and aromatic triad of the Escherichia coli ammonium channel AmtB can be replaced. Proc Natl Acad Sci U S A 108:13270-4
Kim, Kwang-Seo; Pelton, Jeffrey G; Inwood, William B et al. (2010) The Rut pathway for pyrimidine degradation: novel chemistry and toxicity problems. J Bacteriol 192:4089-102
Inwood, William B; Hall, Jason A; Kim, Kwang-Seo et al. (2009) Epistatic effects of the protease/chaperone HflB on some damaged forms of the Escherichia coli ammonium channel AmtB. Genetics 183:1327-40
Inwood, William B; Hall, Jason A; Kim, Kwang-Seo et al. (2009) Genetic evidence for an essential oscillation of transmembrane-spanning segment 5 in the Escherichia coli ammonium channel AmtB. Genetics 183:1341-55
Inwood, William; Yoshihara, Corinne; Zalpuri, Reena et al. (2008) The ultrastructure of a Chlamydomonas reinhardtii mutant strain lacking phytoene synthase resembles that of a colorless alga. Mol Plant 1:925-37
Yoshihara, Corinne; Inoue, Kentaro; Schichnes, Denise et al. (2008) An Rh1-GFP fusion protein is in the cytoplasmic membrane of a white mutant strain of Chlamydomonas reinhardtii. Mol Plant 1:1007-20
Yan, Dalai (2007) Protection of the glutamate pool concentration in enteric bacteria. Proc Natl Acad Sci U S A 104:9475-80

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