that H‐NS promotes DNA interactions in the chromosome at short ranges, but not the long‐range interactions between H‐NS‐regulated genes and operons that were detected by Wang et al. (2011) (Lioy et al. 2018). Work with HiC in Caulobacter has implicated the high‐frequency transcription of long genes as playing a role in boundary formation and maintenance, independent of any effect of translation (Le and Laub 2016). This work suggests that the act of transcribing a long gene inhibits interactions between the DNA regions that flank that gene, causing this long gene to act as a boundary element between domains, a process that may become modulated by H-NS-mediated transcription silencing.
1.43 StpA: A Paralogue of H‐NS
The ratio of H‐NS to its genomic binding sites appears to be important for the competitive fitness of the bacterial cell. Bacteria that gain extra copies of the hns gene through the introduction of multicopy recombinant plasmids that encode this NAP gain in their capacity to replicate themselves (C.J. Dorman, unpublished). The model organisms E. coli and Salmonella typhimurium encode the StpA protein, a second H‐NS‐like molecule. It shares many features with H‐NS and has been described as an RNA chaperone (Doetsch et al. 2010). StpA can substitute for H‐NS and it can form heterodimers with its paralogue (Johansson et al. 2001; Leonard et al. 2009; Sonden and Uhlin 1996; Zhang et al. 1996 Refs). The stpA gene is expressed maximally during exponential growth, perhaps to provide an auxiliary supply of H‐NS‐like protein at a point in the growth cycle where the number of H‐NS binding sites is most numerous (Deighan et al. 2003; Free and Dorman 1997). In this context, it is interesting to note that the transcription of the hns gene is linked positively to chromosome replication (Free and Dorman 1995).
1.44 H‐NS Orthologues Encoded by Plasmids and Phage
Copies of genes encoding H‐NS‐like proteins occur naturally on plasmids, including large, self‐transmissible plasmids (Shintani et al. 2015). These molecules have A+T‐rich DNA and may impose a competitive fitness cost on bacteria that receive them in conjugation by diverting H‐NS from A+T‐rich DNA sites on the chromosome, leading to disruption of the gene expression pattern in the bacterium. By providing its own supply of H‐NS activity, the plasmid can avoid this regulatory disturbance and its associated impact on competitive fitness (Doyle et al. 2007). The R27 plasmid, originally detected in Salmonella, and more recently in S. flexneri 2a 2457T, encodes the H‐NS orthologue Sfh (Beloin et al. 2003a; Deighan et al. 2003). The two proteins exhibit considerable overlaps in their binding sites on the bacterial chromosome. When H‐NS is present, Sfh is restricted to a subset of the sites that it can occupy when H‐NS is removed by inactivation of the hns gene (Dillon et al. 2012). These observations are consistent with Sfh acting as an auxiliary to H‐NS, a role that it shares with StpA. The expression patterns of the three proteins are instructive in this regard: H‐NS is present at a constant level per chromosome throughout the growth cycle, StpA is expressed when the cells are in exponential phase, and Sfh appears at the beginning of stationary phase (Deighan et al. 2003).
H‐NS‐like paralogues encoded by self‐transmissible plasmids (Shintani et al. 2015) and bacteriophage (Skennerton et al. 2011) can downregulate the expression of CRISPR‐cas loci, allowing the mobile genetic element to evade the host immune system (Dillon et al. 2012; Lin et al. 2016; Medina‐Aparicio et al. 2011; Pul et al. 2010). The LysR‐like transcription factor LeuO overcomes H‐NS‐mediated repression of the CRISPR‐cas locus, but the leuO gene is itself silenced by H‐NS, and its paralogues (Dillon et al. 2012; Medina‐Aparicio et al. 2011; Pul et al. 2010). Stochastic upregulation of leuO transcription may provide a mechanism for overcoming silencing of the immunity function in some cells in the population that encounter plasmid or bacteriophage invaders.
1.45 H‐NSB/Hfp and H‐NS2: H‐NS Homologues of HGT Origin
Genes encoding proteins related to H‐NS are found in pathogenicity islands that have been acquired by HGT. Hfp/H‐NSB is an H‐NS‐like protein that is expressed by a gene in the serU pathogenicity island in the chromosome of uropathogenic E. coli (Müller et al. 2010; Williams and Free 2005). It can form heterodimers with H‐NS and may modulate its activity in helping UPEC to adapt to environmental conditions encountered during the infection process (Dorman 2010; Müller et al. 2010). The H‐NSB/Hfp protein is also encoded by a chromosomal island in E. carotovora but is absent from an island in enteropathogenic E. coli (EPEC) that is closely related to the serU island of UPEC (Williams and Free 2005). This association of a gene encoding an H‐NS‐like protein with a former mobile genetic element is reminiscent of similar associations with elements that are currently mobile such as self‐transmissible plasmids and bacteriophage (Section 1.44). The absence of the hnsB gene from the version of the serU island that is present in EPEC is intriguing, given that hnsB is both present and expressed in the corresponding island in UPEC. Perhaps the gene had performed its role once the EPEC island was established in the chromosome and it was subsequently lost in the absence of a selective pressure to keep it? If so, the different environmental circumstances experienced by EPEC and UPEC seem to have selected for retention of hnsB by the latter organism.
Enteroaggregative E. coli (EAEC) strains express, in addition to H‐NS and StpA, an H‐NS2 protein that is closely related to H‐NS. H‐NS2 behaves somewhat like H‐NS when the latter is in a complex with Hha: it targets A+T‐rich genes that have been acquired by HGT and silences them transcriptionally (Prieto et al. 2018). The amino acid sequence of H‐NS2 is similar to those of H‐NSB and Hfp, but differs from them in a number of respects. It does not exhibit the sensitivity to proteolytic turnover that is a characteristic of these H‐NS homologues and StpA (Prieto et al. 2018). It is possible, and plausible, that H‐NS2 and other ‘third homologues’ could form heteromeric complexes with H‐NS or StpA that have distinct activities from those of the homodimers. Certainly, H‐NS heterodimers with StpA have properties that are distinct from those of the homodimers (Johansson et al. 2001; Leonard et al. 2009), so expanding the number of interacting partners may represent a way of modulating NAP function (Beloin et al. ; Sonden and Uhlin 1996; Zhang et al. 1996).
1.46 A Truncated H‐NS‐Like Protein
The serU island in UPEC that encodes H‐NSB/Hfp also encodes a protein that resembles a truncated H‐NS. This is H‐NST and its gene is tightly linked to the hnsB gene in the chromosomal island. H‐NST consists of the first 80 amino acids of H‐NS and the corresponding island in EPEC encodes a closely related protein; EAEC also encodes a relative of H‐NST (Williams and Free 2005). H‐NST from UPEC can form a heterodimer with H‐NS and it can antagonise its activity as a transcription silencer. The corresponding protein from EPEC is much attenuated in its ability to interact with H‐NS and to attenuate its biological activity: a key substitution at residue 16 of the amino acid sequence seems to be responsible for this difference between the UPEC and EPEC H‐NSTs (Williams and Free 2005).
The action of H‐NST recalls that of the gene 5.5 protein that is encoded by bacteriophage T7. Like H‐NST, the gene 5.5 protein co‐purifies with H‐NS and antagonises the transcription silencing activity of H‐NS, presumably to the benefit of the phage (Liu and Richardson 1993). H‐NST and the gene 5.5 protein resemble one another in size and mode of action structure but not in amino acid sequence (Williams and Free 2005). The ability of H‐NST from EPEC to inhibit H‐NS activity has been exploited to explore the H‐NS− phenotype of Yersinia enterocolitica, a bacterium where H‐NS is essential (Baños et al. 2008). The essential nature of H‐NS in Y. enterocolitica probably reflects the absence of a paralogous protein such as StpA that can offset the severe phenotype associated with the loss of H‐NS.
