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The assimilatory pathway of the nitrogen cycle in the haloarchaeon  Haloferax mediterranei  has been well described and characterized in previous studies. However, the regulatory mechanisms involved in the gene expression of this pathway remain unknown in haloarchaea. This work focuses on elucidating the regulation at the transcriptional level of the assimilative  nasABC  operon (HFX_2002 to HFX_2004) through different approaches. Characterization of its promoter region using β-galactosidase as a reporter gene and site-directed mutagenesis has allowed us to identify possible candidate binding regions for a transcriptional factor. The identification of a potential transcriptional regulator related to nitrogen metabolism has become a real challenge due to the lack of information on haloarchaea. The investigation of protein–DNA binding by streptavidin bead pull-down analysis combined with mass spectrometry resulted in the in vitro identification of a transcriptional regulator belonging to the Lrp/AsnC family, which binds to the  nasABC  operon promoter (p. nasABC ). To our knowledge, this study is the first report to suggest the AsnC transcriptional regulator as a powerful candidate to play a regulatory role in  nasABC  gene expression in  Hfx. mediterranei  and, in general, in the assimilatory nitrogen pathway.

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