The Hfq RNA chaperone in the deep-branching Thermotogales lineage: Attempts to reveal its biological role

Bacteria frequently use small RNAs (sRNAs) as part of their regulation of gene expression at the post-transcriptional level. Many of these sRNAs have been shown to depend on the RNA chaperone protein Hfq for their regulatory properties. Homologs of Hfq are found in Archaea and Eukarya, suggesting a common evolutionary predecessor in the last universal common ancestor. The function of Hfq has predominantly been studied in model species and pathogenic bacteria. Yet, no studies have been made on the role of Hfq in deep phylogenetic lineages of bacteria or in thermophiles. The aim of this study was to determine the role of Hfq in gene expression in Thermotogales, a deeply branching and thermophilic lineage. Hfq from Thermosipho africanus (TaHfq) was expressed in Escherichia coli as an insoluble protein and anti-Hfq antibodies were raised in rabbits. Western blot analysis using anti-TaHfq antiserum confirmed that hfq was expressed as a soluble protein in T. africanus. A high degree of conservation in amino acid sequence and protein motifs was confirmed by multiple sequence alignment, and a homology model of TaHfq indicated a similar 3D structure as determined by X-ray crystallography of Hfq from other bacteria, including the presence of a similar RNA-binding pocket. This suggests that Hfq has a role in gene expression in Thermotogales similar to that in model species. This is also supported by the fact that hfq is conserved in the genome-sequences of all the Thermotogales species. Co-immunoprecipitation of Hfq from lysates of T. africanus cells with anti-TaHfq antiserum was performed in order to pull out sRNAs interacting with Hfq, followed by cDNA synthesis. Although the technical procedure appeared functional, it was not possible to isolate cDNA representing sRNAs from T. africanus. This may be the result of a very low expression level of Hfq in T. africanus, as subsequently demonstrated by Proteomics analysis. It is possible that TaHfq is only expressed under certain conditions, such as under stress conditions as found in several other bacteria, or in its natural habitat, where this organism is exposed to a variety of extreme conditions