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

Abstract

Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein–protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σN (σ54) are not conserved in archaea suggesting a novel mechanism of transcriptional control.

Gene Expression of Haloferax volcanii on Intermediate and Abundant Sources of Fixed Nitrogen

“…The PIIs are predicted to regulate transport of ammonium by Amt2 based on analogy to E. coli [37]. H. mediterranei undergoes a similar increase in amt-glnK transcript abundance when starved for nitrogen (N) [27]. Thus, in halophilic archaea, the Amt-transport system is upregulated by shifts to intermediate sources of fixed nitrogen in addition to N-limitation presumably to scavenge ammonium from the environment.…”

“…For instance, genes involved in the assimilatory reduction of nitrate to nitrite ( nasA ) and nitrite to ammonium ( nasD ) are up-regulated in the presence of nitrate [25,26]. In addition, the transcript levels of the high affinity Amt-type ammonia transporter and PII (GlnK) regulators are up during N-starvation in H. mediterranei [27]. The regulatory network responsible for controlling the N-responses in non-methanogenic haloarchaea, however, is poorly understood; sRNAs are implicated in control of the Amt/PII and glutamate dehydrogenase ( gdhA1 ) genes [24], but a master NrpR or NrpA-type transcriptional regulator is not conserved.…”

The structure of a PII signaling protein from a halophilic archaeon reveals novel traits and high‐salt adaptations

“…At present, in the absence of structures in which hmGlnK2 is complexed with 2OG or with its protein partners, it would be speculative to attribute specific T‐loop conformations to given functional states of this halophilic protein. Nevertheless, by analogy with the E. coli PII–AmtB complex , an extended β‐hairpin conformation appears to be likely for the complex demonstrated in H. mediterranei between GlnK and Amt . Given the observations with E. coli , S. elongatus , and A. fulgidus , ADP may promote this extended conformation.…”

Towards the Elucidation of Assimilative nasABC Operon Transcriptional Regulation in Haloferax mediterranei

“…Finally, the construction pVA-p. nas obtained was transformed in Hfx. mediterranei [ 24 , 25 ].…”