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Journal of Bacteriology, February 2002, p. 1028-1040, Vol. 184, No. 4
0021-9193/01/$04.00+0     DOI: 10.1128/jb.184.4.1028-1040.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of GlnK₁ from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK₁

Institut für Mikrobiologie und Genetik, Universität Göttingen, 37077 Göttingen, Germany

Received 30 August 2001/ Accepted 19 November 2001

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.

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