Analysis of Lsm Protein-Mediated Regulation in the Haloarchaeon Haloferax mediterranei.

The Sm protein superfamily includes Sm, like-Sm (Lsm), and Hfq found in the Eukarya, Archaea, and Bacteria domains. Archaeal Lsm proteins have been shown to bind sRNAs and are probably involved in various cellular processes, suggesting a similar function in regulating sRNAs by Hfq in bacteria. Moreover, archaeal Lsm proteins probably represent the ancestral Lsm domain from which eukaryotic Sm proteins have evolved. In this work, Haloferax mediterranei was used as a model organism because it has been widely used to investigate the nitrogen cycle and its regulation in Haloarchaea. Predicting this protein's secondary and tertiary structures has resulted in a three-dimensional model like the solved Lsm protein structure of Archaeoglobus fulgidus. To obtain information on the oligomerization state of the protein, homologous overexpression and purification by means of molecular exclusion chromatography have been performed. The results show that this protein can form hexameric complexes, which can aggregate into 6 or 12 hexameric rings depending on the NaCl concentration and without RNA. In addition, the study of transcriptional expression via microarrays has allowed us to obtain the target genes regulated by the Lsm protein under nutritional stress conditions: nitrogen or carbon starvation. Microarray analysis has shown the first universal stress proteins (USP) in this microorganism that mediate survival in situations of nitrogen deficiency.

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

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 ß-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.