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Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College, Dublin 2, Ireland
Correspondence
Charles J Dorman
(cjdorman{at}tcd.ie)
The H-NS protein has emerged as one of the leading causes of transcriptional repression in Gram-negative bacteria. In a paper published in this issue, De la Cruz and colleagues shed new light on the role of DNA curvature in the repressive mechanism, using a porin gene promoter from the human pathogen Salmonella Typhi as their experimental system (De la Cruz et al., 2009
). Their data help to deepen our understanding of the importance of local DNA structure in facilitating the interaction of H-NS with its target sites in DNA and in establishing an effective nucleoprotein complex for the repression of transcription.
DNA curvature was described as being an important feature of H-NS binding sites many years ago (Yamada et al., 1990) but the reason for its importance remained obscure until recently. This is in spite of the considerable effort that has been expended in analysing H-NS interaction with regions of curvature, using both naturally occurring and artificial sequences with intrinsic curvature (Jordi et al., 1997; Rimsky et al., 2001). A requirement for A+T-rich DNA has also been noted, which is interesting in the light of the facts that (1) A+T-richness is a common feature of bacterial promoters, (2) an appropriate spacing of A+T-rich patches in B-DNA can impose curvature and (3) curved DNA is often found close to promoters (Barbic et al., 2003; Jauregui et al., 2003; Lang et al., 2007). The coincidence of these features seems to produce an ideal platform upon which H-NS can repress the very large number of promoters that it is known to regulate. Central to the repression mechanism is the creation of DNA–H-NS–DNA bridges that impede transcription initiation (Dame et al., 2005; Dorman & Kane, 2009).
De la Cruz et al. (2009) have examined the role of DNA static curvature in the promoter region of the ompS1 porin gene in S. Typhi. Bends were predicted in silico and detected by an electrophoretic technique in which the position of the bend centre relative to the ends of a DNA fragment results in a temperature-dependent alteration in the migration of the DNA molecule through a polyacrylamide gel. Impressively, the authors have been able to remove the bend by making just two changes to the base composition of the DNA, at positions –135 and –151 upstream of the ompS1 transcription start site (+1). The presence of these base substitutions derepresses transcription of the promoter by about sevenfold. H-NS is known to bind to the ompS1 regulatory sequences, as is the paralogous protein StpA (De la Cruz et al., 2007). The previously determined binding sites extend from the region of intrinsic curvature to the transcription start site. In this new study additional binding sites for H-NS are identified upstream of the curve, raising the possibility that H-NS may build bridges between the DNA sequences located upstream and downstream of the curve, resulting in the creation of a nucleoprotein complex that represses the ompS1 promoter. This is an intuitively appealing scenario that is consistent with earlier repression models that involve DNA–H-NS–DNA bridges (Prosseda et al., 2004). Interestingly, the suppression of DNA curvature in the ompS1 upstream regulatory region does not result in full derepression of the promoter; this requires inactivation of the hns gene as well. If the hns gene is inactivated but the curve is left intact (or is restored following mutation) a lower level of derepression results. This hints at the presence in the cell of another curve-dependent repressor of ompS1 transcription. StpA is an attractive candidate, not least because it shares so many properties with H-NS and is known to regulate ompS1 transcription (De la Cruz et al., 2007).
The observation that H-NS continues to exert a negative effect on ompS1 transcription in the absence of the curve suggests that H-NS can still interact with the regulatory region DNA in the absence of bridging across the curve; electrophoretic mobility shift data support this. Of course these data do not rule out the possibility that H-NS may engage in highly localized bridging within each of the ‘arms’ of the regulatory region as well as more conventional binding and nucleation.
The ompS1 promoter is also under the control of other regulators, and the DNA–H-NS–DNA bridging model provides a useful framework in which to consider their contributions at a mechanistic level. Prominent among these are the OmpR and LeuO DNA-binding proteins (De la Cruz et al., 2007; Flores-Valdez et al., 2003; Oropeza et al., 1999). Both are pleiotropic regulators of transcription. OmpR belongs to the response regulator protein family, most of whose members are transcription regulators that are subject to control by phosphorylation by histidine protein kinase sensor proteins (Kenney, 2002). LeuO is a member of the LysR family of DNA-binding proteins (Chen & Wu, 2005; Hernández-Lucas et al., 2008). LeuO is an antagonist of H-NS repression and can act by blocking oligomerization of H-NS along the DNA molecule; it can also form bridges that introduce loops into DNA (Chen & Wu, 2005; Stoebel et al., 2008). Both OmpR and LeuO act positively at the ompS1 promoter (De la Cruz et al., 2007). The data presented by De la Cruz et al. (2009) are consistent with a regulatory mechanism in which alternative DNA interactions associated with LeuO and OmpR, such as DNA bending (OmpR) or the formation of alternative bridged structures (LeuO), diminish the ability of H-NS to maintain the repressive nucleoprotein complex through DNA bridging. When the intrinsic curve upstream of the ompS1 gene is removed by site-directed mutagenesis, the LeuO protein is found to be an even more effective anti-repressor, presumably because its opponent, H-NS, is interacting less strongly with the DNA due to its impeded ability to establish a bridged structure. These findings will assist future investigations of H-NS-repressed promoters in bacteria by helping investigators to make informed predictions of the types of regulatory features they may encounter (i.e. regions of intrinsically curved DNA and H-NS binding sites) and their likely locations relative to the promoter.
ACKNOWLEDGEMENTS
Research in the author's laboratory is supported by grants from Science Foundation Ireland and the Wellcome Trust. I have no conflicts of interest.
REFERENCES
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Related Article
Microbiology 2009 155: 2127-2136.
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