1.20) (Chintakayala et al. 2013). CbpA forms dimers in solution and aggregates following binding to DNA, forming nucleoprotein complexes similar to those produced by the Dps NAP (Cosgriff et al. 2010). The preferred DNA targets of CbpA are A+T‐rich and intrinsically curved; this protein has a marked preference for binding within the Ter macrodomain of the chromosome, a zone of high DNA curvature and with a high A+T content (Chintakayala et al. 2013). CbpA binds at the minor groove of DNA and cbpA mutants display aberrant DNA topology, observations that are consistent with a role in organising the DNA in the Ter macrodomain during stationary phase (Chintakayala et al. 2013, 2015).
Figure 1.20 The stress and stationary phase sigma factor, RpoS. The rpoS gene is influenced at the transcriptional level by several factors. The glucose‐sensitive cAMP‐CRP complex and the stringent‐response signal, ppGpp, act at the RpoD‐dependent promoter to enhance rpoS transcription; production of RpoS is enhanced in mutants deficient in cytosine methylation (see Kahramanoglou et al. 2012). It is important to note that rpoS is expressed under all growth conditions and that the principal regulatory effects are imposed at the level of RpoS protein stability; rapidly growing bacteria have few copies of RpoS and non‐growing bacteria have many. The expansion of the population of RpoS proteins occurs in bacteria when growth is slowed or stopped due to stress. The stress can be physical or chemical in nature. Once transcribed, RpoS mRNA is translated poorly due to the formation of secondary structures that sequester the translation initiation signals. These stem‐loops are eliminated by the DksA sRNA that binds to the 5′ end of the mRNA in the presence of the Hfq RNA chaperone protein. DksA also controls the translation of the hns transcript, albeit negatively due to sequestration of the translation initiation signals. The RpoS protein is degraded by the ClpXP protease. Proteolytic cleavage of RpoS is enhanced by the adaptor protein, RssB. RssB activity is in turn modulated negatively by the sRNAs IraD, IraM, and IraP in response to stresses that impede the growth of the bacterium. In Salmonella, IraM is called RssC. RssB activity is controlled in response to changes to oxygen supply (ArcB) and carbon levels (Acp). The sigma factor must compete with RpoD and other sigma factors for access to the core RNA polymerase and it is assisted in doing so by ppGpp, Crl, and the anti‐sigma factor, Rsd.
Dps is dodecameric in E. coli and has ferritin‐like properties (Grant et al. 1998). This protein accumulates in stationary phase bacteria and was found initially to protect the genomic DNA from oxidative damage (Almirón et al. 1992; Martinez and Kolter 1997). It does not impede transcription, despite being an abundant DNA‐binding protein (Janissen et al. 2018). Dps was subsequently discovered also to afford protection against gamma radiation, ultraviolet light, copper and iron toxicity, heat stress, and pH shock (Algu et al. 2007; Jeong et al. 2008; Nair and Finkel 2004). It also protects DNA from cleavage by restriction enzymes (Janissen et al. 2018).
Although Dps is usually grouped with the NAPs (Ali Azam and Ishihama 1999), its relationship with DNA has been difficult to determine with precision. This protein can form a co‐crystal with DNA, perhaps accounting for its ability to protect the chromosome from damage in stressed cells (Wolf et al. 1999). Through the application of SELEX (systematic evolution of ligands by exponential enrichment) to E. coli, a DNA sequence has been identified that seems to contain the elements of a Dps‐binding site (Ishihama et al. 2016). A closely related motif has been detected in E. coli by chromatin immunoprecipitation on chip (Antipov et al. 2017). These Dps‐binding sites overlap with those of other NAPs, leading to speculation that Dps supplies the genome architectural functions of those proteins (such as FIS) that are no longer expressed as the bacterium enters stationary phase (Antipov et al. 2017). Alternatively, Dps binding and the binding of other NAPs, such as IHF, may alternate depending on the environmental conditions that accompany entry of the bacterium into stationary phase (Lee et al. 2015).
The FIS protein plays a key role in controlling the expression of the dps gene during the growth cycle (Grainger et al. 2008). This gene is transcribed from a single promoter that is recognised by both the RpoD‐ and the RpoS‐containing forms of RNA polymerase (Altuvia et al. 1994). Three NAPs are involved in the regulation of dps transcription: FIS, H‐NS, and IHF. IHF and the OxyR transcription factor activate the dps promoter in association with RpoD in bacteria experiencing oxidative stress; in stationary phase the same promoter is utilised by RNA polymerase containing RpoS (Altuvia et al. 1994). During exponential growth, FIS holds RNA polymerase containing RpoD at the promoter while H‐NS excludes the RpoD form of the RNA polymerase holoenzyme from the same promoter. The transcriptionally inert FIS‐RpoD‐RNA‐polymerase complex prevents entry of the RpoS‐containing RNA polymerase holoenzyme. Since FIS levels decline to negligible values as the bacterium enters stationary phase, this negative control is no longer exerted, and as H‐NS is unable to impede the activity of the RpoS‐containing form of RNA polymerase, dps transcription can begin (Grainger et al. 2008).
1.42 The H‐NS Protein: A Silencer of Transcription
Throughout the 1980s, the gene that encodes the H‐NS NAP was discovered and rediscovered by investigators working independently of each other because this protein is involved in controlling the expression of so many different components of the bacterium. One of the consequences of the broad influence of H‐NS is that the gene that encodes it has been given many names, such as bglY, osmZ, pilG, virR, and dxdR, among others. In each case, the name linked a mutation in the gene, now referred to universally as hns, to a specific H‐NS‐dependent system in the cell such as beta‐glucoside uptake and utilisation (bglY), pilus expression (pilG), the osmotic stress response (osmZ), expression of a virulent phenotype in the pathogen Shigella (virR), or thermo‐regulated adhesin expression in E. coli (drdX) (Defez and de Felice 1981; Higgins et al. 1988; Göransson et al. 1990; Maurelli and Sansonetti 1988; Spears et al. 1986). The hns gene is located in the Ter macrodomain of the chromosome, close to the topA gene, and early experiments revealed a connection between some hns alleles and alterations in the DNA topology of reporter plasmids in those strains (Dorman et al. 1990; Higgins et al. 1988). The finding that H‐NS binding to DNA impedes access to that DNA by DNA gyrase may explain these observations (Sutormin et al. 2019). H‐NS can influence transposition and site‐specific recombination as well as transcription (Corcoran and Dorman 2009; Liu et al. 2011; Whitfield et al. 2009) and it has effects at the level of mRNA translation too (Park et al. 2010).
The H‐NS protein is a small, abundant NAP that is produced at all stages of the growth cycle (Ali Azam and Ishihama 1999; Dorman 2013; Free and Dorman 1995). It binds to A+T‐rich DNA and has been described as having a preference for DNA with intrinsic curvature (Yamada et al. 1991). These features are commonly associated with transcriptional promoters, allowing H‐NS to target large numbers of genes for repression, even if the functions of the gene products are not related in any obvious way. The protein is almost always a repressor, allowing it to silence transcription of a large subset of the genes in the genome (Figure 1.21).
Figure 1.21 The vast H‐NS regulon. The H‐NS protein controls the expression of hundreds of genes in the pan genome, the accessory genome, and the core genome. Its own gene is also subject to complex control at the transcriptional and posttranscriptional levels. Among the factors influencing hns transcription positively are chromosome replication, the growth‐phase‐dependent FIS protein, and the cold‐shock regulatory protein CspA; it is auto‐repressed by its own gene product and repressed by the iron‐binding Fur protein, and translation of H‐NS's own mRNA is inhibited by the DsrA sRNA in an Hfq‐dependent manner. H‐NS,
