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.
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