3.2) Hfq, a nucleoid-associated protein
Hfq has also been described as one of the NAP that shape the bacterial chromosome (Azam and Ishihama, 1999). Hfq is capable of binding double-stranded and single-stranded DNA but with less affinity than RNA (Updegrove et al. , 2010; Geinguenaud et al. , 2011; Kubiaket al. , 2022). The majority of studies examining Hfq as a NAP have been conducted in E. coli . Fluorescence microscopy imaging of single DNA molecules and atomic force microscopy experiments have demonstrated the formation of a nucleoprotein complex between Hfq and double-stranded DNA (Jiang et al. , 2015). The nucleoprotein complex between Hfq and DNA remains flexible with a moderate increase in bending persistence length, compared to the rigid filaments observed after H-NS binding (Boudreau et al. , 2018). However, as observed for H-NS, the nucleoprotein complex with DNA compacts the DNA into a condensed form by bridging DNA segments (Jiang et al. , 2015). The DNA binding involves the CTR domain (Updegrove et al. , 2010), a sequence of 35 amino acids that has been predicted to be unstructured, but whose crystal structure remains unknown (Vogel and Luisi, 2011). Upon interaction with DNA, these domains self-assemble and form amyloid-like fibrillar structures in vitro (Arluison et al. , 2006; Fortas et al. , 2015). These structures have been shown to be responsible for the self-assembly with DNA, the DNA bridging and compaction (Figure 1 ) (Malabirade et al. , 2018a). Although the full-length Hfq binds to DNA via the interface of its toroidal hexameric ring, the NTR by itself is not required for Hfq to bind to DNA and only the presence of the CTR is necessary for DNA compaction (Malabirade et al. , 2017). A model has been proposed whereby Hfq can form a bridge by anchoring one or more of its other CTR arms to another section of the same or another DNA molecule and/or by CTR-mediated self-interactions among multiple proteins (Malabiradeet al. , 2017). Recently, it was confirmed that amyloid structures are formed in vivo (Partouche et al. , 2019), indicating that the CTR is responsible for the nucleoid remodelling in vivo through DNA binding, bridging and compaction (Figure 1 ) (Cossaet al. , 2022). In contrast to H-NS, the bridging formed by the nucleoprotein complex doesn’t affect DNA topology (Malabirade et al. , 2018a). It has therefore been suggested that the effect of Hfq on DNA supercoiling observed in vivo (Tsui, Leung and Winkler, 1994) may be indirect. Indeed, Hfq post-transcriptionally regulates the expression of proteins that affect DNA topology (Figure 1 ) (Sledjeski, Whitman and Zhang, 2001). Finally, the CTR of Hfq has been shown to bind to G-quadruplexes, a type of alternative DNA and RNA structure. G-quadruplex structures consist of three or more guanine quadruplex rings that are held together by Hoogsteen hydrogen bonds (Gellert, Lipsett and Davies, 1962), forming highly stable four-stranded structures. Hfq enhances the stability of G-quadruplex structures, which can lead to the termination of DNA replication and a significant increase in the mutation rate. Therefore, the stabilisation of G-quadruplexes by Hfq may drive the evolution or alternation of bacterial gene expression (Parekh et al. , 2019, 2020).