In vitroproof of direct regulation of glutamine synthetase by GlnK proteins in the extreme halophilic archaeonHaloferax mediterranei

Abstract

Haloferax mediterranei is an extreme halophilic micro-organism belonging to the Archaea domain that was isolated from the Santa Pola solar salterns (Alicante, Spain) in 1983. The biochemistry of the proteins involved in nitrogen metabolism is being studied, but the knowledge of their regulation is very scarce at present. The PII superfamily is constituted by major regulators of nitrogen metabolism, which are widespread in prokaryotic and eukaryotic organisms. These trimeric proteins (12 kDa per subunit) have in Escherichia coli long been known to regulate GS (glutamine synthetase) activity via its adenylyltransferase/adenylyl-removing enzyme and, more recently, to be able to interact directly with this enzyme in methanogenic archaea. We have tested the possible role of PII proteins in the regulation of ammonium assimilation in our model organism and the results clearly indicate that the direct influence of GS by PII proteins can also take place in halophilic archaea, starting with the comprehension of nitrogen regulation in those organisms.

Nitrogen regulation of protein–protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea

“…The levels of amtB‐glnK transcripts agree with our previous study in which GlnK levels were only detected by anti‐GlnK immunoblot under nitrate‐rich conditions when cells were starved for ammonium (Pedro‐Roig et al. ). The a mtB1‐glnK1 and amtB2‐glnK2 probes of this study share DNA sequence homology, making it difficult to assess whether both gene pairs are transcribed.…”

“…This nitrogen‐regulated expression was in good agreement with the profiles observed for glnK1‐amtB1 transcript (see above) and GlnK protein (Pedro‐Roig et al. ). The results suggested that AmtB1 could be produced in Hfx.…”

“…) and agrees with our previous analysis of GlnK expression regulated by nitrogen at the protein level (Pedro‐Roig et al. ). In Gram‐negative bacteria such as E. coli and Pseudomonas putida , glnK‐amtB is similarly regulated by nitrogen availability with expression silent in the presence of ammonium and high under conditions of ammonium starvation (Atkinson et al.…”

“…For GlnK detection, customized anti‐GlnK polyclonal rabbit antibodies (Pedro‐Roig et al. ) were used in combination with a peroxidase‐labeled secondary antibody and the chemiluminescent substrate luminol (BM Chemiluminescence Western Blotting Kit from Roche). A commercial anti‐Flag antibody (Monoclonal ANTI‐FLAG M2‐Alkaline Phosphatase Clone M2 from Sigma) and CDP‐Star Chemiluminescent Substrate (Applied Biosystems, Foster City, CA) were used for Flag‐AmtB identification.…”

“…; Pedro‐Roig et al. ). PII proteins regulate the assimilation of nitrogen compounds by interacting noncovalently with target proteins (Chellamuthu et al.…”