Characterization of GlnK1 from Methanosarcina mazei strain Gö1: complementation of an Escherichia coli glnK mutant strain by GlnK1.
Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1-amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.
Ehlers
Claudia
C
Institut für Mikrobiologie und Genetik, Universität Göttingen, 37077 Göttingen, Germany.
Grabbe
Roman
R
Veit
Katharina
K
Schmitz
Ruth A
RA
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics; D027682:Cation Transport Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005816:Genetic Complementation Test; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000254:growth & development; D017020:Methanosarcina / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009584:Nitrogen / Q000378:metabolism; D009586:Nitrogen Fixation; D009876:Operon; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
C111096:AmtB protein, E coli; D019843:Archaeal Proteins; D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; C449645:GlnK1 protein, Methanosarcina mazei; D009584:Nitrogen