Biochemical Characterization of Novel Hfq-like Proteins in Borg Extrachromosomal Elements

Borgs are giant, linear extrachromosomal elements found in anaerobic methane-oxidizing archaea. They feature pervasive tandem repeat (TR) regions that undergo rapid evolution, encoding new TRs within open reading frames and their subsequent proteins. Here, we report the identification of a monophyletic group of novel Hfq-like proteins in Borgs genomes. Hfq is a pleiotropic regulator of bacterial gene expression, aiding small RNAs to bind with their mRNA targets. Based on phylogenetic and structural bioinformatics, Borg Hfq appears to form hexameric complexes. Strikingly, Borg Hfqs display amino acid tandem repeats in their disordered region, which is located at the N-terminal domain (NTD) compared to the C-terminal domain in bacterial Hfq. We find through biochemical analysis that bacterial and Borg Hfqs display similar RNA binding properties. In-vivo growth experiments show successful rescue of E. coli Hfq knockout strains by plasmids with Borg Hfqs. Furthermore, NTD-deleted Borg Hfq did not show any negative impact on RNA binding or growth rescue, indicating that the NTD is not necessary for its function. Overall, this study provides insight into the diversity and evolution of Hfq-like proteins and highlights their potential applications in genetic engineering and regulatory processes in microorganisms.