Ammonia assimilation by Ruminococcus flavefaciens FD-1
Duncan, Paul Alan
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Permalink
https://hdl.handle.net/2142/22186
Description
Title
Ammonia assimilation by Ruminococcus flavefaciens FD-1
Author(s)
Duncan, Paul Alan
Issue Date
1993
Doctoral Committee Chair(s)
Mackie, Roderick I.
Department of Study
Animal Sciences
Discipline
Animal Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Agriculture, Animal Culture and Nutrition
Language
eng
Abstract
Studies were conducted to better understand the mechanism by which Ruminococcus flavefaciens FD-1 utilizes ammonia (-um). $\sp{14}$C-methyl-ammonium was not taken up by R. flavefaciens FD-1, but cells prepared similarly depleted ammonia from media (initial rates of 11.2 and 0.8 nmol$\cdot$min$\sp{-1}{\cdot}$mg$\sp{-1}$ protein for cells grown with limiting nitrogen and carbon, respectively).
Glutamine synthetase activity was only detected using the $\gamma$-glutamyl transferase assay. The specific activity was 10-fold higher when cells were grown in ammonia-limiting compared to carbon-limiting defined medium (574 versus 60 nmol$\cdot$min$\sp{-1}{\cdot}$mg$\sp{-1}$ for whole cell assays). Specific activity was reduced by 10-30% by sudden exposure of cells grown with ammonia-limitation to high levels of ammonia, but this loss of activity was not consistent with the adenylylation model of post-translational regulation found in the enteric bacteria.
Glutamate dehydrogenase activity was 3-fold higher in cells grown in ammonia-limiting compared to carbon-limiting defined medium. Glutamate dehydrogenase was purified 119-fold from batch grown cells. The K$\sb{\rm m}$'s for ammonia, $\alpha$-ketoglutarate, and glutamate were 19.2, 0.41 and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The native enzyme and subunits are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The first residue in the amino-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified post-translationally. Comparison of the amino-terminal sequence with those of E. coli, S. typhimurium and Clostridium symbiosum revealed only 39% amino acid homologies.
A degenerate oligonucleotide probe was synthesized and used to screen a recombinant subgenomic library of R. flavefaciens FD-1 chromosomal DNA for the gene encoding glutamate dehydrogenase.
Ammonium uptake rates were 1-2 orders of magnitude lower than GS $\gamma$GT specific activity, and 2-3 orders of magnitude lower than that of GDH. This suggests that the enzymatic incorporation of ammonia into amino acid form (or at least glutamate) is not a limiting step in ammonia assimilation.
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