Supercoiling is the superhelical tension adopted by chromosomal or plasmid DNA, a process regulated by the balancing activities of two enzymes, topoisomerase I and the DNA gyrase (topoisomerase II). In bacteria, changes in the cellular ATP/ADP balance, environmental osmolarity, temperature and anaerobiosis are among the factors known to affect DNA supercoiling and subsequently alter gene expression. Changes in these physiological and environmental conditions are also among those one might expect bacteria to encounter when moving from one niche to another. The activity of some bacterial promoters responding to temperature changes is known to correlate with the degree of DNA supercoiling, and therefore supercoiling is considered to be one link between environmental changes and gene expression. Supercoiling has emerged as a central actor in many temperature-regulated virulence regulons (Dorman, 1991).
Uropathogenic Escherichia coli produce Pap pili when grown at 37°C but not at 25°C, indicating that the pap locus is thermoregulated (Göransson and Uhlin, 1984). Pili production is controlled by the transcription activator PapI, whose transcript papI was shown to be limiting at lower temperatures and to accumulate at a thermal upshift to 37°C. Overproduction of papI mRNA leads to a loss in thermoregulation. Later, papI was shown to be derepressed in a Δhns strain, implicating the H-NS protein in the repression of pap genes at lower temperatures (Göransson et al., 1990). H-NS is a histone-like protein with the ability to affect DNA supercoiling and is involved in condensing the bacterial chromosome into a chromatin-like structure, influencing processes such as recombination, transposition and transcription (Higgins et al., 1990; Hulton et al., 1990). Thus, the authors suggested that temperature causes changes in the DNA topology around the pap locus and thereby influences the binding and repressing ability of H-NS, hence the temperature regulation (Göransson et al., 1990). More recent studies demonstrated that in vitro H-NS blocks methylation of pap DNA (methylation is essential to pap regulation) in a protein concentration-dependent manner, and that in vivo, below physiological temperature, the presence of H-NS leads to methylation protection that represses transcription (White-Ziegler et al., 1998). The authors speculated that temperature directly affects H-NS and compromises its ability to productively repress DNA (White-Ziegler et al., 1998). A situation parallel to the pap system exists in Shigella, in which the virulence loci are regulated by the VirF regulator that activates virB transcription leading, in turn, to activation of the target effector genes (Tobe et al., 1991). In experiments in which virF mRNA was overproduced at 30°C virB could not be activated, whereas in a Δhns strain virB was constitutively active, suggesting that H-NS (VirR in Shigella) negatively regulates this process (Dorman et al., 1990; Tobe et al., 1993). Also, in vitro footprinting and transcription assays showed that H-NS binds a region in the virB promoter, probably blocking the action of RNA polymerase and thus acts as a repressor, whereas VirF binds to an upstream sequence and activates transcription but only from supercoiled DNA (Tobe et al., 1993). Interestingly, experiments on overproduction of H-NS at 37°C show that it is able to act as repressor at that temperature in contrast to the wild-type situation (where less H-NS is produced) and led to a proposal that it is the amount of H-NS that is the key to regulation (Hromockyj et al., 1992). In this proposed scenario, the levels of H-NS dictate its state of oligomerization that determines the ability to act as a repressor (Hromockyj et al., 1992). Yet another pathogenic bacterium, Yersinia enterocolitica seems to use changes in DNA topology as a regulatory means. In Y. enterocolitica chromosomally encoded YmoA is a histone-like protein with properties similar to H-NS but no shared similarity at the amino acid sequence level (Mikulskis and Cornelis, 1994). YmoA seems to be a ‘global’ repressor of the yop virulence regulon in Yersinia, implicating supercoiling as one putative mechanism of control as in the above examples (Lambert de Rouvroit et al., 1992). One study proposes that elevated temperature, which has been shown to affect reporter plasmid supercoiling, would dislodge YmoA and allow the VirF activator in Y. enterocolitica (not to be confused with Shigella VirF) to activate genes (Rohde et al., 1994).
Given the above examples, how does supercoiling affect promoter activity and what is the temperature sensor in this process? There are several candidate scenarios, some of which may be acting in concert. It is quite plausible that the ability of an effector protein, such as H-NS, to dock on the nucleic acid target is influenced by a temperature-mediated change in the topology of that particular stretch of DNA. Alternatively, H-NS may itself respond to temperature and release bound DNA, which can then be altered with respect to supercoiling. In-depth structure–function analysis is needed to address the question of whether H-NS is alone able to sense temperature. H-NS sensing could proceed by ways such as direct temperature-induced conformational changes or varying the state of oligomerization, both leading to the altered ability to bind DNA. Perhaps both DNA topology and H-NS conformation (which would dictate the ability to bind) are both temperature sensitive. Also, topoisomerase I, DNA gyrase and proteins capable of binding to curved DNA (other than H-NS) may act as direct sensors (although there is no evidence to this effect) or via factors that regulate them, complicating the issue even further. In this scenario, DNA topology would be modulated according to the environmental conditions, and factors such as H-NS would be secondary mediators in this regulatory scheme. It is tempting to speculate that all paths would converge on a protein(s) that receives and transmits the thermal cue.