Characterization of GlnK(1) from Methanosarcina mazei strain Go1: Complementation of an Escherichia coli glnK mutant strain by GlnK(1)

Abstract

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 Gol and show that H. mazei GlnK(1) 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 GlnK(1) is encoded in the glnK(1)-amtB(1) 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 glnK(1) shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK(1) was cloned and overexpressed in E. coli, and GlnK(1) was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK(1)-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 GlnK(1) indicated significant differences compared to E. coli GlnK. First, GlnK(1) from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK(1) and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK(1) 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.