GENETIC STUDIES OF GLUTAMINE SYNTHETASE IN BACTERIA

Summary

Principal Investigator: SYDNEY GOVONS KUSTU
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: ----------------1986 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc A previously undescribed pathway for pyrimidine catabolism
    Kevin D Loh
    Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720 3102, USA
    Proc Natl Acad Sci U S A 103:5114-9. 2006
  2. pmc The molecular basis of K+ exclusion by the Escherichia coli ammonium channel AmtB
    Jason A Hall
    Division of Biological Sciences, University of California San Diego, La Jolla, California 92093 0374, USA
    J Biol Chem 288:14080-6. 2013
  3. pmc Need-based activation of ammonium uptake in Escherichia coli
    Minsu Kim
    Department of Physics, University of California at San Diego, La Jolla, CA, USA
    Mol Syst Biol 8:616. 2012
  4. pmc An Rh1-GFP fusion protein is in the cytoplasmic membrane of a white mutant strain of Chlamydomonas reinhardtii
    Corinne Yoshihara
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 3102, USA
    Mol Plant 1:1007-20. 2008
  5. pmc The ultrastructure of a Chlamydomonas reinhardtii mutant strain lacking phytoene synthase resembles that of a colorless alga
    William Inwood
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 3102, USA
    Mol Plant 1:925-37. 2008
  6. pmc The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion
    Rebecca N Fong
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 3102, USA
    Proc Natl Acad Sci U S A 104:18706-11. 2007
  7. pmc Protection of the glutamate pool concentration in enteric bacteria
    Dalai Yan
    Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202 5120, USA
    Proc Natl Acad Sci U S A 104:9475-80. 2007
  8. pmc Natural history of transposition in the green alga Chlamydomonas reinhardtii: use of the AMT4 locus as an experimental system
    Kwang Seo Kim
    Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA
    Genetics 173:2005-19. 2006
  9. ncbi Biological gas channels for NH3 and CO2: evidence that Rh (Rhesus) proteins are CO2 channels
    S Kustu
    Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, Berkeley, CA 94720 3102, USA
    Transfus Clin Biol 13:103-10. 2006
  10. pmc Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12
    Jason Vo
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
    Proc Natl Acad Sci U S A 110:8696-701. 2013

Scientific Experts

  • Jason A Hall
  • Sydney Kustu
  • Kwang Seo Kim
  • William Inwood
  • William B Inwood
  • Corinne Yoshihara
  • Dalai Yan
  • Jason Vo
  • Minsu Kim
  • Rebecca N Fong
  • Kevin D Loh
  • S Kustu
  • John M Hayes
  • Alexander Groisman
  • Terence Hwa
  • Zhongge Zhang
  • Hiroyuki Okano
  • Jeffrey G Pelton
  • Ulla Andersen
  • David E Wemmer
  • Lusine Demirkhanyan
  • Helen Zgurskaya
  • David Wemmer
  • Denise Schichnes
  • Kentaro Inoue
  • Steven Ruzin
  • Reena Zalpuri
  • W Inwood
  • Rebecca Fong
  • Prasad Gyaneshwar
  • Rebecca Parales
  • Zhongrui Zhou
  • Eirene Markenscoff Papadimitriou

Detail Information

Publications13

  1. pmc A previously undescribed pathway for pyrimidine catabolism
    Kevin D Loh
    Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720 3102, USA
    Proc Natl Acad Sci U S A 103:5114-9. 2006
    ..The product of the divergently transcribed gene, b1013, is a tetracycline repressor family regulator that controls transcription of the b1012 operon negatively...
  2. pmc The molecular basis of K+ exclusion by the Escherichia coli ammonium channel AmtB
    Jason A Hall
    Division of Biological Sciences, University of California San Diego, La Jolla, California 92093 0374, USA
    J Biol Chem 288:14080-6. 2013
    ....
  3. pmc Need-based activation of ammonium uptake in Escherichia coli
    Minsu Kim
    Department of Physics, University of California at San Diego, La Jolla, CA, USA
    Mol Syst Biol 8:616. 2012
    ..This novel strategy ensures that the expensive cost of upholding the internal ammonium concentration against back diffusion is kept at a minimum...
  4. pmc An Rh1-GFP fusion protein is in the cytoplasmic membrane of a white mutant strain of Chlamydomonas reinhardtii
    Corinne Yoshihara
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 3102, USA
    Mol Plant 1:1007-20. 2008
    ....
  5. pmc The ultrastructure of a Chlamydomonas reinhardtii mutant strain lacking phytoene synthase resembles that of a colorless alga
    William Inwood
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 3102, USA
    Mol Plant 1:925-37. 2008
    ..The ultrastructure of lts1 mutant strains is similar to but distinct from that of previously described white and yellow mutant strains of C. reinhardtii and is similar to that of naturally colorless algae of the Polytoma group...
  6. pmc The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion
    Rebecca N Fong
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 3102, USA
    Proc Natl Acad Sci U S A 104:18706-11. 2007
    ..We address the role of W148 in decreasing the activity and increasing the selectivity of AmtB and the implications of our findings with respect to the function of Rh proteins, the only known homologues of Amt/Mep proteins...
  7. pmc Protection of the glutamate pool concentration in enteric bacteria
    Dalai Yan
    Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202 5120, USA
    Proc Natl Acad Sci U S A 104:9475-80. 2007
    ..Analysis of other suppressor mutants selected on solid medium suggests that increased GDH expression is the key for rescue of the growth defect of GOGAT(-) mutants under low NH(4)(+) conditions...
  8. pmc Natural history of transposition in the green alga Chlamydomonas reinhardtii: use of the AMT4 locus as an experimental system
    Kwang Seo Kim
    Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA
    Genetics 173:2005-19. 2006
    ..The sequence of the other and bioinformatic analysis indicates that it derives from a miniature retrotransposon or TRIM, which we propose to call MRC1 (miniature retrotransposon of Chlamydomonas)...
  9. ncbi Biological gas channels for NH3 and CO2: evidence that Rh (Rhesus) proteins are CO2 channels
    S Kustu
    Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, Berkeley, CA 94720 3102, USA
    Transfus Clin Biol 13:103-10. 2006
    ....
  10. pmc Mechanism for nitrogen isotope fractionation during ammonium assimilation by Escherichia coli K12
    Jason Vo
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
    Proc Natl Acad Sci U S A 110:8696-701. 2013
    ..9‰). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them...
  11. pmc Epistatic effects of the protease/chaperone HflB on some damaged forms of the Escherichia coli ammonium channel AmtB
    William B Inwood
    Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA
    Genetics 183:1327-40. 2009
    ..HflB apparently "tacks" damaged AmtB tails to the adjacent monomers. Knowing that HflB has intervened is prerequisite to determining the functional basis for AmtB inactivation...
  12. pmc The Rut pathway for pyrimidine degradation: novel chemistry and toxicity problems
    Kwang Seo Kim
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 3102, USA
    J Bacteriol 192:4089-102. 2010
    ..The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter...
  13. pmc The pivotal twin histidines and aromatic triad of the Escherichia coli ammonium channel AmtB can be replaced
    Jason A Hall
    Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720 3102, USA
    Proc Natl Acad Sci U S A 108:13270-4. 2011
    ..Our findings lead us to conclude that these elements and their component parts are not required for AmtB function, but instead serve to optimize its performance...