title: The glutamine synthetase of Prevotella bryantii B(1)4 is a family III enzyme (GlnN) and glutamine supports growth of mutants lacking glutamate dehydrogenase activity.
abstract: Prevotella spp. are believed to play a central role in ruminal nitrogen metabolism, but little is understood about the genetics and biochemistry of nitrogen assimilation and regulation in these bacteria. The gene encoding a family III glutamine synthetase (GSIII, glnN) in Prevotella bryantii B(1)4 was cloned by Escherichia coli mutant complementation, and enzyme assays as well as Northern blot analysis showed that maximal enzyme activity and glnN transcription occurred in cells grown under nitrogen-limiting conditions. Addition of methionine sulfoximine (MSX), a GS inhibitor, terminated bacterial growth when ammonium was provided as the sole nitrogen source, but the inhibitory effect could be overcome by the inclusion of either L-glutamine or trypticase in the growth medium. A P. bryantii mutant lacking glutamate dehydrogenase (GdhA) activity was isolated by ethylmethylsulfonate mutagenesis. Growth studies with different nitrogen sources showed that the mutant strain was still capable of growth with ammonium as the sole nitrogen source, albeit at a decreased growth rate. The mutant strain could also grow with L-glutamine as a nitrogen source in the presence of MSX. These data suggest that GlnN provides an effective route of ammonium assimilation for P. bryantii, in addition to that afforded by the glutamate dehydrogenase pathway.
authors.affiliation: Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE 68583-0908, USA. zwen@dental.ufl.edu
authors.forename: Zezhang T
authors.initials: ZT
authors.lastname: Wen
authors.forename: Lansha
authors.initials: L
authors.lastname: Peng
authors.forename: Mark
authors.initials: M
authors.lastname: Morrison
mesh_terms: D000595:Amino Acid Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D005973:Glutamine / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018720:Prevotella / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D012333:RNA, Messenger; D005973:Glutamine; D005969:Glutamate Dehydrogenase; D005974:Glutamate-Ammonia Ligase