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Silencing of foreign DNA in bacteria
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Silencing of foreign DNA in bacteria Sabrina S Ali1, Bin Xia2, Jun Liu1 and William Wiley Navarre1 Xenogeneic silencing proteins facilitate horizontal gene transfer by silencing expression of AT-rich sequences. By virtue of their activity these proteins serve as master regulators of a variety of important functions including motility, drug resistance, and virulence. Three families of silencers have been identified to date: the H-NS like proteins of Gram-negative bacteria, the MvaT like proteins of Pseudomonacae, and the Lsr2 proteins of Actinobacteria. Structural and biochemical characterization of these proteins have revealed that they share surprising commonalities in mechanism and function despite extensive divergence in both sequence and structure. Here we discuss the mechanisms that underlie the ability of these proteins to selectively target AT-rich DNA and the contradictory data regarding the mode by which H-NS forms nucleoprotein complexes. Addresses 1 Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada 2 Beijing NMR Center, School of Life Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Corresponding author: Navarre, William Wiley ([email protected])
Current Opinion in Microbiology 2012, 15:175–181 This review comes from a themed issue on Cell Regulation Edited by Vanessa Sperandio and Nancy E Freitag Available online 20th January 2012 1369-5274/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2011.12.014
Introduction Continually faced with changing environmental conditions, free-living bacteria are under enormous pressure to develop new traits and gain a selective advantage over competing strains. Crucial to bacterial adaptation and survival is the ability to obtain new genetic material via horizontal gene transfer (HGT). HGT, which has been described as ‘evolution in quantum leaps’, allows bacteria to rapidly acquire novel functions such as the ability to survive within eukaryotic host cells, combat other bacteria, and gain resistance to antimicrobial agents [1–4]. Despite their potential benefits, however, newly acquired sequences are particularly problematic from a regulatory point of view [5]. Among other things, the inappropriate expression of a foreign gene can result in a significant fitness cost for the recipient cell. For example, one study www.sciencedirect.com
that examined difficult to clone sequences during the preparation of genomic libraries determined that high levels of expression, presumably resulting in toxicity, pose a significant barrier to the successful transfer of new sequences [6]. The genomes of different bacterial species can range in overall GC-content from less than 20% to as much as 75%. However within a given genome the GC-content of most genes are kept relatively uniform and close to that of the genome average such that the GC-content and codon usage patterns can form a signature of a particular phylogenetic group. Accordingly, DNA that has been acquired from a foreign source (xenogeneic DNA) can often be distinguished from the ancestral chromosome based on its deviation from the pattern of codon usage and nucleotide composition present in its new host [7–9]. It was noted from early genomic analysis that xenogeneic sequences frequently display higher adenine and thymine (AT) content compared to the host genome [10], but the underlying cause of this bias is unclear. A potential benefit of having a characteristic ‘pattern’ with regard to codon bias and GC-content is the ability to distinguish self DNA from non-self DNA. Several bacterial species produce proteins that specifically identify and silence expression from xenogeneic DNA sequences that possess lower GC-content than the host genome. These proteins, which we term ‘xenogeneic silencers’, include the H-NS family of proteins from several Gram-negative bacteria as well as the MvaT and MvaU paralogs from Pseudomonas and the Lsr2 proteins from Mycobacteria [11]. As a result of their activity these silencing proteins serve as master regulators of many functions associated with virulence, drug resistance, and ancillary metabolic pathways [12,13]. In Salmonella, for example, H-NS binds to and downregulates the expression of over 12% of the Salmonella genome including all five pathogenicity islands [14,15]. MvaT/MvaU and Lsr2 also target AT-rich regions in the genomes of Pseudomaonas and Mycobacteria, respectively [16,17]. Xenogeneic silencers ameliorate the detrimental consequences of HGT while facilitating the integration of horizontally acquired genes into pre-existing regulatory networks [18]. This assertion is supported by the findings that loss of H-NS or MvaT/MvaU are highly detrimental for cell growth, albeit to varying degrees in different strains. Improved fitness of Salmonella mutants can be obtained via secondary deletions in genomic islands or their regulators [14,15]. Current Opinion in Microbiology 2012, 15:175–181
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Common properties of xenogeneic silencers The three families of xenogeneic silencing proteins identified to date (H-NS like, MvaT like and Lsr2) are dissimilar in sequence but all have the ability to selectively bind AT-rich DNA and all require cooperative binding in order to downregulate gene expression. Structurally, the most well understood of the xenogeneic silencers is H-NS of E. coli/Salmonella, a small 15 kDa protein with a modular architecture consisting of three distinct domains: an N-terminal dimerization domain (residues 1–64); a central secondary dimerization domain (residues 64–80), and a C-terminal DNA binding domain (residues 80–137) [19,20,21]. MvaT and Lsr2 share a similar overall domain organization to H-NS where the region involved in dimerization and the formation of higher order oligomers is found within an N-terminal domain while DNA binding activity is contained within the C-terminal end of the molecule [17,22].
How do xenogeneic silencers target AT-rich DNA? One question is how silencers specifically target AT-rich DNA without recognizing a specific sequence. The apparent affinity of H-NS for a ‘genuine’ target sequence differs from that of non-specific DNA by less than an order of magnitude and as a result of this H-NS can completely coat a strand of DNA if it is present at high enough concentrations [23,24]. Early studies of H-NS affinity measured the cooperative H-NS binding to larger sequences, however, and subtleties in the mechanism by which individual H-NS molecules bind DNA were obscure. In a more recent detailed study of the proV promoter region revealed that H-NS can bind some sites with surprisingly high affinity (Kd 15 nM; consensus: tCG(t/a)T(a/t)AATT) [25]. Binding at such high affinity sites likely initiates the cooperative binding of other HNS molecules to proximal AT-rich sites of lower affinity [26]. Combined with H-NS’s fast off rates of DNA binding, this unconventional ‘bind and spread’ model likely enables H-NS to form dynamic complexes along DNA targets, a feature that may facilitate subsequent countersilencing [27,28]. High-resolution docking models of H-NS and Lsr2 binding to model DNA sequences have been achieved based on binding titration data using two-dimensional 1H–15N heteronuclear single-quantum correlation (HSQC) experiments [17,29]. Remarkably these studies revealed that both Lsr2 and H-NS employ a common mechanism to selectively target AT-rich DNA (Figure 1). The H-NS family of proteins is defined by a consensus motif within the C-terminal DNA binding domain (TWTGX1GX2X3P) where the residues at positions X1 are most frequently arginine or glutamine and residues at position X2 are typically arginine or lysine. In H-NS of E. coli and Salmonella the X1 residue is glutamine and the X2 residue is arginine. Docking models from NMR titration Current Opinion in Microbiology 2012, 15:175–181
Figure 1
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(b)
Current Opinion in Microbiology
H-NS and Lsr2 bind AT-rich DNA via a conserved prokaryotic AT-hook motif. The docking models of (a) H-NS (blue) and (b) Lsr2 (gold) with model DNA substrates [17,29]. The C-terminal domains of H-NS and Lsr2 are dissimilar in their tertiary structure but share a common mechanism of DNA binding. Highlighted in pink are the side chains of residues Q112, G113 and R114 of H-NS and R97, G98 and R99 of Lsr2. These residues are located within loop regions of both structures. The side chains of the QGR (H-NS) and RGR (Lsr2) motifs are shown interacting with the minor groove of a DNA substrate, as determined by HSQC experiments. The binding motif and mechanism employed by HNS and Lsr2 are similar to how the eukaryotic HMG-1 proteins target ATrich DNA via an Arg-Gly-Arg containing AT-hook motif. This mechanism of binding also explains the high affinity of H-NS for the proV motif, where the distorted minor groove of the TpA step was found to be a critical binding determinant [73].
experiments indicate that this QGR motif inserts into the minor groove of AT-rich DNA such that the side chains of glutamine and arginine extend in opposite directions [29]. Similar studies that modeled the interaction of Lsr2 with target sequences identified a conserved RGR motif similar to the QGR motif of H-NS that inserts into the minor groove of DNA target sequences [17]. Both H-NS and Lsr2 binding of DNA is reminiscent of how the abundant eukaryotic HMG-I(Y) proteins interact with target DNA via an ‘AT-hook’ motif [30–32]. In agreement with the AT-hook model are further observations that mutations of either X1 or X2 residue to alanine in both H-NS and Lsr2 abolish binding, and that both proteins are effectively competed for binding by drugs that intercalate into the minor groove like netropsin and distamycin [29,33]. Binding of AT-rich DNA via a ‘prokaryotic AT-hook’ motif can explain how xenogeneic silencers selectively target AT-rich DNA over mixed-base and GC-rich DNA. The minor groove of AT-rich DNA lacks the exocyclic 6NH2 group present in GC base pairs and AT-rich DNA can therefore accommodate deeper intrusions of protein side chains into the groove. AT- rich DNA sequences also adopt a minor groove that is significantly narrower than DNA of mixed GC/AT-content or GC-rich DNA. Atracts, defined as at least three consecutive ApA, TpT or ApT steps, narrow the minor groove due to negative propeller twisting (deviating from co-planarity) of base www.sciencedirect.com
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pairs that is stabilized by inter-base pair hydrogen bonds in the major groove [34,35]. The narrow minor groove in ATrich DNA leads to a surface that has considerably more electronegative potential than is found in mixed-base or GC-rich DNA [36–38]. The TpA step, on the other hand, does not allow the formation of an A-tract like conformation due to steric clash between the cross-strand adenines [39,40]. Instead the TpA step causes increased flexibility of the DNA and significant widening of the minor groove. The presence of a slightly wider point in the minor groove at a TpA step likely enables prokaryotic AT-hooks to penetrate deeply within the minor groove where the residues in the X1 and X2 position are positioned in opposite orientations to interact with the narrow negatively charged backbone of the DNA. The structures of MvaT or MvaU have not been solved but they appear to lack an AThook motif and the mechanism by which they target ATrich DNA may be distinct from that of H-NS and Lsr2.
Higher order oligomerization as the basis of cooperative binding Early studies on H-NS revealed that it binds DNA in a cooperative manner and DNaseI footprinting assays of several model promoters demonstrated that H-NS binding protects large stretches of (AT-rich) DNA [24,41]. These findings suggested that H-NS forms higher order nucleoprotein complexes along target sequences, an assertion further supported in biophysical studies discussed further below [42–44]. The importance of higher order oligomerization for silencing has been demonstrated through mutational analysis for both H-NS and MvaT, where it was demonstrated that mutant proteins that lack the ability to form higher order oligomers are defective for silencing even though they retain their ability to bind DNA [22,23,45]. This is further supported by findings that silencing can be antagonized by proteins that alter the ability of H-NS to properly oligomerize [46,47]. H-NS, MvaT and Lsr2 can spontaneously oligomerize in solution in the absence of DNA [22,45,48]. For H-NS this oligomerization is concentration dependent and studies have variously reported the oligomeric state of H-NS in solution, ranging from a dimer at 10 mM to 20mers at higher concentrations (344 mM) [45,49–51]. The molecular details of how H-NS self-associates were recently clarified by Arold et al. who solved the structure of the S. typhimurium H-NS from residues 1–83 in an oligomerized state [19]. In agreement with previous solution structures from E. coli [52] and S. Typhimurium [20], the N-terminal dimerization domain of H-NS is composed of two short alpha helixes followed by an elongated third helix (H3). The recent structure revealed the presence of a short fourth helix (H4) that forms a secondary dimerization site with the distal end of H3 by means of a helix– turn–helix motif between residues 57–83. Through simultaneous dimerization at both interfaces, H-NS filaments can propagate in a ‘head-to-head/tail-to-tail’ manner and www.sciencedirect.com
create a ‘superhelical scaffold’ (Figure 2). Notably, multimerization of MvaT was also show require two distinct domains that may function in a fashion analogous to the two dimerization domains in H-NS [22]. An early controversy with regard to H-NS, whether the dimer arranged in a parallel or antiparallel fashion [20,52,53], seems to have been resolved with the recent structure convincingly showing that monomers in an H-NS dimer arrange in an antiparallel orientation. A controversial DNA binding property of H-NS is its ability to bridge adjacent DNA helices, a property that is shared with Lsr2 and MvaT [48,54–56]. H-NS induced DNA bridges were initially discovered by atomic force microscopy imaging [54–56] and these bridging complexes were further studied by Dame et al. by means of a novel quantum trap device called a ‘Q-Trap’ [27]. In this study the ends of two individual DNA molecules were each tethered to polystyrene beads that could then be individually manipulated by four independent optical traps. This enabled measurements of the force required to unzip bridged DNA–H-NS–DNA complexes. The data from these experiments were interpreted to indicate that H-NS binds DNA predominantly as a dimer with bridging being mediated when the binding domains within the dimer crosslink adjacent DNA strands. The recent structure by Arold et al., provides a different view where the H-NS superhelical scaffold structure orients the DNA binding domains of adjacent monomers on opposing faces along the superhelix [19]. This arrangement could in theory accommodate the binding of two DNA helices per H-NS scaffold, which would provide a structural rationale for how bridging occurs (Figure 2b). Bridging by H-NS has been invoked to explain its ability to constrain DNA supercoiling states [57], and induce loops and bending within DNA strands [58]. The bridging mode of binding is also attractive in its ability to explain why silencing of bgl and proV loci requires cooperative interactions between H-NS binding sites both upstream and downstream of the promoter [59]. The notion that bridge formation is the relevant DNA binding mode of H-NS in vivo has been challenged by studies of H-NS/DNA interactions using magnetic tweezers. In contrast to the compact bridged structures observed by Dame et al., Amit et al. noted that the addition of H-NS to l-DNA induced an end-to-end DNA extension [60]. The extended H-NS/DNA complexes were reportedly more rigid than naked DNA and have been referred to as a ‘stiffening mode’ of DNA binding. In an attempt to rectify these contradicting reports, Liu et al. recently proposed that H-NS binds DNA in bridging mode only when in the presence of magnesium ions at concentrations of 5 mM or higher whereas H-NS binds in stiffening mode at physiologically relevant Mg2+ concentrations [61], but the exact manner by which Mg2+ affects the switch is unclear. In further Current Opinion in Microbiology 2012, 15:175–181
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Figure 2
(a) DNA Binding Domain
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DNA BINDING DOMAINS ON OPPOSITE FACES (BRIDGING)
DNA BINDING DOMAINS ON SAME FACE Current Opinion in Microbiology
Oligomerization of H-NS through two dimerization interfaces. (a) Schematic diagram of H-NS monomers arranged in their multimerized state via a ‘head-to-head/tail-to-tail’ mechanism. Helices H1, H2 and H3 form the N-terminal dimerization interface. The central dimerization domain is formed by a helix–turn–helix motif formed between the C-terminal end of H3 and H4, which is shown between the yellow and blue H-NS monomers. (b) Two modes of DNA binding by H-NS have been proposed: bridging and stiffening. H-NS may engage in bridging if the DNA binding domains are present on opposing faces of the helical structure. If, however, the DNA binding domains rotate in such a way that they bind a single DNA molecule then stiffening may occur. (c) A three-dimensional representation of the extended helical filament formed by 16 H-NS dimers as determined by Arold et al. [19]. The DNA binding domains are not shown.
support of the stiffening model, the transcription factor SsrB that activates the H-NS repressed SPI-2 locus could displace H-NS from DNA when bound in ‘stiffening mode’, but not when H-NS–DNA bridges were present [62]. It is difficult to envision a scenario where both bridging and stiffening modes of binding are physiologically relevant. With regard to what we know about H-NS structure it is possible that stiffening results when DNA binding domains align along the same face of the extended HNS filament (Figure 2). If the stiffening mode of binding is relevant in vivo it is possible that binding of rigid H-NS filaments upstream and downstream of promoters, as is the Current Opinion in Microbiology 2012, 15:175–181
case at proV and bgl, occludes RNA polymerase or prevents the formation of an open complex, especially if the filament can span across the unbound region. MvaT, Lsr2, and the H-NS paralog StpA, each of which can functionally complement H-NS in vivo [63,64], have all been found to bridge DNA [48,54], and whether these molecules can also bind DNA in the stiffening mode has yet to be tested.
How does H-NS downregulate transcription? Remarkably another point of controversy is how H-NS binding functions to downregulate transcription. Several distinct mechanisms of H-NS silencing have been proposed at different promoters, which are conceivably consistent with either the stiffening or bridging mode of www.sciencedirect.com
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DNA binding. Some promoters sensitive to local topology may be locked in an inactive state due to the ability of H-NS to constrain supercoils [65]. H-NS binding at some promoters may occlude core promoter elements from binding by RNAP. At the rrnB promoter H-NS blocks transcription apparently by trapping it in an open complex but preventing elongation [66]. Two studies that tried to address whether trapping of RNAP by H-NS is the predominant mechanism of silencing in vivo using chromatin immunoprecipitation came to opposite conclusions, where one study suggested trapping is common at many promoters (based on H-NS/ RNAP co-occupancy) while the other study suggested H-NS occludes RNAP at most promoters [14,67]. It has been proposed that polymerase trapping is due to the formation of a repressory loop between H-NS linked regions [68,69]. Indeed a ‘trapped’ RNAP complex at the hdeAB promoter that involved DNA bridging by H-NS was visualized by atomic force microscopy [69]. In this case, RNAP in complex with the housekeeping sigma factor s70 (RNAPs70) facilitated the formation of H-NS bridges whereas RNAPs38 did not. If the stiffening model of DNA binding prevails these data will have to be revisited with more in depth mechanistic studies to determine why RNAPs38 is able to counter H-NS-mediated repression at promoters where RNAPs70 is blocked. Regardless, an understanding of the mechanism by which H-NS prevents transcription is critical to understanding how H-NS can be antagonized to activate transcription of foreign genes under physiologically appropriate conditions. Excellent reviews on diverse mechanisms of countersilencing have recently been written [5,11,28].
under physiologically appropriate conditions. The use of high-resolution visualization and single molecule biophysical approaches have yielded important insights but have also provided contradictory data. It is important that any hypothesis generated from biophysical data be congruent with genetic, high-throughput, and biochemical data generated by more traditional approaches.
Where to go from here
1.
Dobrindt U, Chowdary MG, Krumbholz G, Hacker J: Genome dynamics and its impact on evolution of Escherichia coli. Med Microbiol Immunol 2010, 199:145-154.
2.
Hacker J, Kaper JB: Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol 2000, 54:641-679.
3.
Ochman H, Lawrence JG, Groisman EA: Lateral gene transfer and the nature of bacterial innovation. Nature 2000, 405:299-304.
4.
Groisman EA, Ochman H: Pathogenicity islands: bacterial evolution in quantum leaps. Cell 1996, 87:791-794.
The recent advances in understanding the mechanism of how xenogeneic silencers bind DNA reveals that they target and repress foreign sequences based on their topological differences from the host chromosome. A recent study of chromatin structure in live cells suggesting that H-NS regulated genes may form distinct topological clusters distinct from the remainder of the chromosome, a finding that may also support bridging or other forms high-order association by H-NS in vivo [70]. We still, however, lack a clear mechanistic understanding of how H-NS binding effectively downregulates gene expression and how it assembles into a nucleoprotein structure that can be selectively antagonized under the appropriate conditions. We also lack any understanding of the role accessory molecules like Hha and YdgT play in silencing by H-NS [71,72]. The barriers to progress are due in part to the fact that H-NS has several unique properties that make it more challenging than ‘typical’ DNA binding proteins to study. Future progress will require the use of multiple complementary experimental approaches including well-controlled in vitro transcription assays and biophysical measurements www.sciencedirect.com
It is remarkable that Lsr2 and H-NS, which bear no similarity either in sequence or overall structure, and coming from species separated by over two billion years of evolution, have independently arrived at a common mechanism to target AT-rich DNA. Additional insights may still come through the comparative analysis of H-NS to functional analogs from other species. It is likely that the ability of silencers from different families to complement one another in vivo means their structures in the context of a nucleoprotein filament (either by bridging or stiffening) will be similar as well.
Acknowledgements The Navarre laboratory is supported by an Operating Grant and New Investigator Award from the Canada Institutes for Health Research (MOP86683 and MSH-87729) and a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN 386286-10). JL is supported by a CIHR Operating Grant (MOP-15107) and BX by Grant No. 2009CB521703 from the 973 Program of China. SSA is supported by a graduate fellowship from the Natural Sciences and Engineering Research Council of Canada.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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62. Walthers D, Li Y, Liu Y, Anand G, Yan J, Kenney LJ: Salmonella enterica response regulator SsrB relieves H-NS silencing by displacing H-NS bound in polymerization mode and directly activates transcription. J Biol Chem 2011, 286:1895-1902. Three studies that suggest bridging is not the relvant mode of DNA binding by H-NS.
48. Chen JM, Ren H, Shaw JE, Wang YJ, Li M, Leung AS, Tran V, Berbenetz NM, Kocincova D, Yip CM et al.: Lsr2 of Mycobacterium tuberculosis is a DNA-bridging protein. Nucleic Acids Res 2008, 36:2123-2135. 49. Falconi M, Gualtieri MT, La Teana A, Losso MA, Pon CL: Proteins from the prokaryotic nucleoid: primary and quaternary structure of the 15-kD Escherichia coli DNA binding protein HNS. Mol Microbiol 1988, 2:323-329. 50. Smyth CP, Lundback T, Renzoni D, Siligardi G, Beavil R, Layton M, Sidebotham JM, Hinton JC, Driscoll PC, Higgins CF et al.: Oligomerization of the chromatin-structuring protein H-NS. Mol Microbiol 2000, 36:962-972. 51. Spurio R, Falconi M, Brandi A, Pon CL, Gualerzi CO: The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending. EMBO J 1997, 16:1795-1805. 52. Bloch V, Yang Y, Margeat E, Chavanieu A, Auge MT, Robert B, Arold S, Rimsky S, Kochoyan M: The H-NS dimerization domain defines a new fold contributing to DNA recognition. Nat Struct Biol 2003, 10:212-218. 53. Cerdan R, Bloch V, Yang Y, Bertin P, Dumas C, Rimsky S, Kochoyan M, Arold ST: Crystal structure of the N-terminal dimerisation domain of VicH, the H-NS-like protein of Vibrio cholerae. J Mol Biol 2003, 334:179-185. 54. Dame RT, Luijsterburg MS, Krin E, Bertin PN, Wagner R, Wuite GJ: DNA bridging: a property shared among H-NS-like proteins. J Bacteriol 2005, 187:1845-1848. 55. Dame RT, Wyman C, Goosen N: H-NS mediated compaction of DNA visualised by atomic force microscopy. Nucleic Acids Res 2000, 28:3504-3510. 56. Dame RT, Wyman C, Goosen N: Structural basis for preferential binding of H-NS to curved DNA. Biochimie 2001, 83:231-234.
63. Tendeng C, Soutourina OA, Danchin A, Bertin PN: MvaT proteins in Pseudomonas spp.: a novel class of H-NS-like proteins. Microbiology 2003, 149:3047-3050. 64. Gordon BR, Imperial R, Wang L, Navarre WW, Liu J: Lsr2 of Mycobacterium represents a novel class of H-NS-like proteins. J Bacteriol 2008, 190:7052-7059. 65. Blot N, Mavathur R, Geertz M, Travers A, Muskhelishvili G: Homeostatic regulation of supercoiling sensitivity coordinates transcription of the bacterial genome. EMBO Rep 2006, 7:710-715. 66. Schroder O, Wagner R: The bacterial DNA-binding protein H-NS represses ribosomal RNA transcription by trapping RNA polymerase in the initiation complex. J Mol Biol 2000, 298:737-748. 67. Grainger DC, Hurd D, Goldberg MD, Busby SJ: Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome. Nucleic Acids Res 2006, 34:4642-4652. 68. Dame RT, Wyman C, Wurm R, Wagner R, Goosen N: Structural basis for H-NS-mediated trapping of RNA polymerase in the open initiation complex at the rrnB P1. J Biol Chem 2002, 277:2146-2150. 69. Shin M, Song M, Rhee JH, Hong Y, Kim YJ, Seok YJ, Ha KS, Jung SH, Choy HE: DNA looping-mediated repression by histone-like protein H-NS: specific requirement of Esigma70 as a cofactor for looping. Genes Dev 2005, 19:2388-2398. 70. Wang W, Li GW, Chen C, Xie XS, Zhuang X: Chromosome organization by a nucleoid-associated protein in live bacteria. Science 2011, 333:1445-1449.
57. Hardy CD, Cozzarelli NR: A genetic selection for supercoiling mutants of Escherichia coli reveals proteins implicated in chromosome structure. Mol Microbiol 2005, 57:1636-1652.
71. Silphaduang U, Mascarenhas M, Karmali M, Coombes BK: Repression of intracellular virulence factors in Salmonella by the Hha and YdgT nucleoid-associated proteins. J Bacteriol 2007, 189:3669-3673.
58. Dame RT, van Mameren J, Luijsterburg MS, Mysiak ME, Janicijevic A, Pazdzior G, van der Vliet PC, Wyman C, Wuite GJ: Analysis of scanning force microscopy images of proteininduced DNA bending using simulations. Nucleic Acids Res 2005, 33:e68.
72. Cordeiro TN, Garci AJ, Pons JI, Aznar S, Juarez A, Pons M: A single residue mutation in Hha preserving structure and binding to H-NS results in loss of H-NS mediated gene repression properties. FEBS Lett 2008, 582:3139-3144.
59. Nagarajavel V, Madhusudan S, Dole S, Rahmouni AR, Schnetz K: Repression by binding of H-NS within the transcription unit. J Biol Chem 2007, 282:23622-23630. 60. Amit R, Oppenheim AB, Stavans J: Increased bending rigidity of single DNA molecules by H-NS, a temperature and osmolarity sensor. Biophys J 2003, 84:2467-2473.
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73. Sette M, Spurio R, Trotta E, Brandizi C, Brandi A, Pon CL, Barbato G, Boelens R, Gualerzi CO: Sequence-specific recognition of DNA by the C-terminal domain of nucleoidassociated protein H-NS. J Biol Chem 2009, 284:30453-30462. This study provides insight into the critical determinants of DNA binding by H-NS and why the TpA step is an important feature of high-affinity binding sites.
Current Opinion in Microbiology 2012, 15:175–181
Silencing of foreign DNA in bacteria Sabrina S Ali1, Bin Xia2, Jun Liu1 and William Wiley Navarre1 Xenogeneic silencing proteins facilitate horizontal gene transfer by silencing expression of AT-rich sequences. By virtue of their activity these proteins serve as master regulators of a variety of important functions including motility, drug resistance, and virulence. Three families of silencers have been identified to date: the H-NS like proteins of Gram-negative bacteria, the MvaT like proteins of Pseudomonacae, and the Lsr2 proteins of Actinobacteria. Structural and biochemical characterization of these proteins have revealed that they share surprising commonalities in mechanism and function despite extensive divergence in both sequence and structure. Here we discuss the mechanisms that underlie the ability of these proteins to selectively target AT-rich DNA and the contradictory data regarding the mode by which H-NS forms nucleoprotein complexes. Addresses 1 Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada 2 Beijing NMR Center, School of Life Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Corresponding author: Navarre, William Wiley ([email protected])
Current Opinion in Microbiology 2012, 15:175–181 This review comes from a themed issue on Cell Regulation Edited by Vanessa Sperandio and Nancy E Freitag Available online 20th January 2012 1369-5274/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2011.12.014
Introduction Continually faced with changing environmental conditions, free-living bacteria are under enormous pressure to develop new traits and gain a selective advantage over competing strains. Crucial to bacterial adaptation and survival is the ability to obtain new genetic material via horizontal gene transfer (HGT). HGT, which has been described as ‘evolution in quantum leaps’, allows bacteria to rapidly acquire novel functions such as the ability to survive within eukaryotic host cells, combat other bacteria, and gain resistance to antimicrobial agents [1–4]. Despite their potential benefits, however, newly acquired sequences are particularly problematic from a regulatory point of view [5]. Among other things, the inappropriate expression of a foreign gene can result in a significant fitness cost for the recipient cell. For example, one study www.sciencedirect.com
that examined difficult to clone sequences during the preparation of genomic libraries determined that high levels of expression, presumably resulting in toxicity, pose a significant barrier to the successful transfer of new sequences [6]. The genomes of different bacterial species can range in overall GC-content from less than 20% to as much as 75%. However within a given genome the GC-content of most genes are kept relatively uniform and close to that of the genome average such that the GC-content and codon usage patterns can form a signature of a particular phylogenetic group. Accordingly, DNA that has been acquired from a foreign source (xenogeneic DNA) can often be distinguished from the ancestral chromosome based on its deviation from the pattern of codon usage and nucleotide composition present in its new host [7–9]. It was noted from early genomic analysis that xenogeneic sequences frequently display higher adenine and thymine (AT) content compared to the host genome [10], but the underlying cause of this bias is unclear. A potential benefit of having a characteristic ‘pattern’ with regard to codon bias and GC-content is the ability to distinguish self DNA from non-self DNA. Several bacterial species produce proteins that specifically identify and silence expression from xenogeneic DNA sequences that possess lower GC-content than the host genome. These proteins, which we term ‘xenogeneic silencers’, include the H-NS family of proteins from several Gram-negative bacteria as well as the MvaT and MvaU paralogs from Pseudomonas and the Lsr2 proteins from Mycobacteria [11]. As a result of their activity these silencing proteins serve as master regulators of many functions associated with virulence, drug resistance, and ancillary metabolic pathways [12,13]. In Salmonella, for example, H-NS binds to and downregulates the expression of over 12% of the Salmonella genome including all five pathogenicity islands [14,15]. MvaT/MvaU and Lsr2 also target AT-rich regions in the genomes of Pseudomaonas and Mycobacteria, respectively [16,17]. Xenogeneic silencers ameliorate the detrimental consequences of HGT while facilitating the integration of horizontally acquired genes into pre-existing regulatory networks [18]. This assertion is supported by the findings that loss of H-NS or MvaT/MvaU are highly detrimental for cell growth, albeit to varying degrees in different strains. Improved fitness of Salmonella mutants can be obtained via secondary deletions in genomic islands or their regulators [14,15]. Current Opinion in Microbiology 2012, 15:175–181
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Common properties of xenogeneic silencers The three families of xenogeneic silencing proteins identified to date (H-NS like, MvaT like and Lsr2) are dissimilar in sequence but all have the ability to selectively bind AT-rich DNA and all require cooperative binding in order to downregulate gene expression. Structurally, the most well understood of the xenogeneic silencers is H-NS of E. coli/Salmonella, a small 15 kDa protein with a modular architecture consisting of three distinct domains: an N-terminal dimerization domain (residues 1–64); a central secondary dimerization domain (residues 64–80), and a C-terminal DNA binding domain (residues 80–137) [19,20,21]. MvaT and Lsr2 share a similar overall domain organization to H-NS where the region involved in dimerization and the formation of higher order oligomers is found within an N-terminal domain while DNA binding activity is contained within the C-terminal end of the molecule [17,22].
How do xenogeneic silencers target AT-rich DNA? One question is how silencers specifically target AT-rich DNA without recognizing a specific sequence. The apparent affinity of H-NS for a ‘genuine’ target sequence differs from that of non-specific DNA by less than an order of magnitude and as a result of this H-NS can completely coat a strand of DNA if it is present at high enough concentrations [23,24]. Early studies of H-NS affinity measured the cooperative H-NS binding to larger sequences, however, and subtleties in the mechanism by which individual H-NS molecules bind DNA were obscure. In a more recent detailed study of the proV promoter region revealed that H-NS can bind some sites with surprisingly high affinity (Kd 15 nM; consensus: tCG(t/a)T(a/t)AATT) [25]. Binding at such high affinity sites likely initiates the cooperative binding of other HNS molecules to proximal AT-rich sites of lower affinity [26]. Combined with H-NS’s fast off rates of DNA binding, this unconventional ‘bind and spread’ model likely enables H-NS to form dynamic complexes along DNA targets, a feature that may facilitate subsequent countersilencing [27,28]. High-resolution docking models of H-NS and Lsr2 binding to model DNA sequences have been achieved based on binding titration data using two-dimensional 1H–15N heteronuclear single-quantum correlation (HSQC) experiments [17,29]. Remarkably these studies revealed that both Lsr2 and H-NS employ a common mechanism to selectively target AT-rich DNA (Figure 1). The H-NS family of proteins is defined by a consensus motif within the C-terminal DNA binding domain (TWTGX1GX2X3P) where the residues at positions X1 are most frequently arginine or glutamine and residues at position X2 are typically arginine or lysine. In H-NS of E. coli and Salmonella the X1 residue is glutamine and the X2 residue is arginine. Docking models from NMR titration Current Opinion in Microbiology 2012, 15:175–181
Figure 1
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(b)
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H-NS and Lsr2 bind AT-rich DNA via a conserved prokaryotic AT-hook motif. The docking models of (a) H-NS (blue) and (b) Lsr2 (gold) with model DNA substrates [17,29]. The C-terminal domains of H-NS and Lsr2 are dissimilar in their tertiary structure but share a common mechanism of DNA binding. Highlighted in pink are the side chains of residues Q112, G113 and R114 of H-NS and R97, G98 and R99 of Lsr2. These residues are located within loop regions of both structures. The side chains of the QGR (H-NS) and RGR (Lsr2) motifs are shown interacting with the minor groove of a DNA substrate, as determined by HSQC experiments. The binding motif and mechanism employed by HNS and Lsr2 are similar to how the eukaryotic HMG-1 proteins target ATrich DNA via an Arg-Gly-Arg containing AT-hook motif. This mechanism of binding also explains the high affinity of H-NS for the proV motif, where the distorted minor groove of the TpA step was found to be a critical binding determinant [73].
experiments indicate that this QGR motif inserts into the minor groove of AT-rich DNA such that the side chains of glutamine and arginine extend in opposite directions [29]. Similar studies that modeled the interaction of Lsr2 with target sequences identified a conserved RGR motif similar to the QGR motif of H-NS that inserts into the minor groove of DNA target sequences [17]. Both H-NS and Lsr2 binding of DNA is reminiscent of how the abundant eukaryotic HMG-I(Y) proteins interact with target DNA via an ‘AT-hook’ motif [30–32]. In agreement with the AT-hook model are further observations that mutations of either X1 or X2 residue to alanine in both H-NS and Lsr2 abolish binding, and that both proteins are effectively competed for binding by drugs that intercalate into the minor groove like netropsin and distamycin [29,33]. Binding of AT-rich DNA via a ‘prokaryotic AT-hook’ motif can explain how xenogeneic silencers selectively target AT-rich DNA over mixed-base and GC-rich DNA. The minor groove of AT-rich DNA lacks the exocyclic 6NH2 group present in GC base pairs and AT-rich DNA can therefore accommodate deeper intrusions of protein side chains into the groove. AT- rich DNA sequences also adopt a minor groove that is significantly narrower than DNA of mixed GC/AT-content or GC-rich DNA. Atracts, defined as at least three consecutive ApA, TpT or ApT steps, narrow the minor groove due to negative propeller twisting (deviating from co-planarity) of base www.sciencedirect.com
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pairs that is stabilized by inter-base pair hydrogen bonds in the major groove [34,35]. The narrow minor groove in ATrich DNA leads to a surface that has considerably more electronegative potential than is found in mixed-base or GC-rich DNA [36–38]. The TpA step, on the other hand, does not allow the formation of an A-tract like conformation due to steric clash between the cross-strand adenines [39,40]. Instead the TpA step causes increased flexibility of the DNA and significant widening of the minor groove. The presence of a slightly wider point in the minor groove at a TpA step likely enables prokaryotic AT-hooks to penetrate deeply within the minor groove where the residues in the X1 and X2 position are positioned in opposite orientations to interact with the narrow negatively charged backbone of the DNA. The structures of MvaT or MvaU have not been solved but they appear to lack an AThook motif and the mechanism by which they target ATrich DNA may be distinct from that of H-NS and Lsr2.
Higher order oligomerization as the basis of cooperative binding Early studies on H-NS revealed that it binds DNA in a cooperative manner and DNaseI footprinting assays of several model promoters demonstrated that H-NS binding protects large stretches of (AT-rich) DNA [24,41]. These findings suggested that H-NS forms higher order nucleoprotein complexes along target sequences, an assertion further supported in biophysical studies discussed further below [42–44]. The importance of higher order oligomerization for silencing has been demonstrated through mutational analysis for both H-NS and MvaT, where it was demonstrated that mutant proteins that lack the ability to form higher order oligomers are defective for silencing even though they retain their ability to bind DNA [22,23,45]. This is further supported by findings that silencing can be antagonized by proteins that alter the ability of H-NS to properly oligomerize [46,47]. H-NS, MvaT and Lsr2 can spontaneously oligomerize in solution in the absence of DNA [22,45,48]. For H-NS this oligomerization is concentration dependent and studies have variously reported the oligomeric state of H-NS in solution, ranging from a dimer at 10 mM to 20mers at higher concentrations (344 mM) [45,49–51]. The molecular details of how H-NS self-associates were recently clarified by Arold et al. who solved the structure of the S. typhimurium H-NS from residues 1–83 in an oligomerized state [19]. In agreement with previous solution structures from E. coli [52] and S. Typhimurium [20], the N-terminal dimerization domain of H-NS is composed of two short alpha helixes followed by an elongated third helix (H3). The recent structure revealed the presence of a short fourth helix (H4) that forms a secondary dimerization site with the distal end of H3 by means of a helix– turn–helix motif between residues 57–83. Through simultaneous dimerization at both interfaces, H-NS filaments can propagate in a ‘head-to-head/tail-to-tail’ manner and www.sciencedirect.com
create a ‘superhelical scaffold’ (Figure 2). Notably, multimerization of MvaT was also show require two distinct domains that may function in a fashion analogous to the two dimerization domains in H-NS [22]. An early controversy with regard to H-NS, whether the dimer arranged in a parallel or antiparallel fashion [20,52,53], seems to have been resolved with the recent structure convincingly showing that monomers in an H-NS dimer arrange in an antiparallel orientation. A controversial DNA binding property of H-NS is its ability to bridge adjacent DNA helices, a property that is shared with Lsr2 and MvaT [48,54–56]. H-NS induced DNA bridges were initially discovered by atomic force microscopy imaging [54–56] and these bridging complexes were further studied by Dame et al. by means of a novel quantum trap device called a ‘Q-Trap’ [27]. In this study the ends of two individual DNA molecules were each tethered to polystyrene beads that could then be individually manipulated by four independent optical traps. This enabled measurements of the force required to unzip bridged DNA–H-NS–DNA complexes. The data from these experiments were interpreted to indicate that H-NS binds DNA predominantly as a dimer with bridging being mediated when the binding domains within the dimer crosslink adjacent DNA strands. The recent structure by Arold et al., provides a different view where the H-NS superhelical scaffold structure orients the DNA binding domains of adjacent monomers on opposing faces along the superhelix [19]. This arrangement could in theory accommodate the binding of two DNA helices per H-NS scaffold, which would provide a structural rationale for how bridging occurs (Figure 2b). Bridging by H-NS has been invoked to explain its ability to constrain DNA supercoiling states [57], and induce loops and bending within DNA strands [58]. The bridging mode of binding is also attractive in its ability to explain why silencing of bgl and proV loci requires cooperative interactions between H-NS binding sites both upstream and downstream of the promoter [59]. The notion that bridge formation is the relevant DNA binding mode of H-NS in vivo has been challenged by studies of H-NS/DNA interactions using magnetic tweezers. In contrast to the compact bridged structures observed by Dame et al., Amit et al. noted that the addition of H-NS to l-DNA induced an end-to-end DNA extension [60]. The extended H-NS/DNA complexes were reportedly more rigid than naked DNA and have been referred to as a ‘stiffening mode’ of DNA binding. In an attempt to rectify these contradicting reports, Liu et al. recently proposed that H-NS binds DNA in bridging mode only when in the presence of magnesium ions at concentrations of 5 mM or higher whereas H-NS binds in stiffening mode at physiologically relevant Mg2+ concentrations [61], but the exact manner by which Mg2+ affects the switch is unclear. In further Current Opinion in Microbiology 2012, 15:175–181
178 Cell Regulation
Figure 2
(a) DNA Binding Domain
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H4 H4
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DNA BINDING DOMAINS ON OPPOSITE FACES (BRIDGING)
DNA BINDING DOMAINS ON SAME FACE Current Opinion in Microbiology
Oligomerization of H-NS through two dimerization interfaces. (a) Schematic diagram of H-NS monomers arranged in their multimerized state via a ‘head-to-head/tail-to-tail’ mechanism. Helices H1, H2 and H3 form the N-terminal dimerization interface. The central dimerization domain is formed by a helix–turn–helix motif formed between the C-terminal end of H3 and H4, which is shown between the yellow and blue H-NS monomers. (b) Two modes of DNA binding by H-NS have been proposed: bridging and stiffening. H-NS may engage in bridging if the DNA binding domains are present on opposing faces of the helical structure. If, however, the DNA binding domains rotate in such a way that they bind a single DNA molecule then stiffening may occur. (c) A three-dimensional representation of the extended helical filament formed by 16 H-NS dimers as determined by Arold et al. [19]. The DNA binding domains are not shown.
support of the stiffening model, the transcription factor SsrB that activates the H-NS repressed SPI-2 locus could displace H-NS from DNA when bound in ‘stiffening mode’, but not when H-NS–DNA bridges were present [62]. It is difficult to envision a scenario where both bridging and stiffening modes of binding are physiologically relevant. With regard to what we know about H-NS structure it is possible that stiffening results when DNA binding domains align along the same face of the extended HNS filament (Figure 2). If the stiffening mode of binding is relevant in vivo it is possible that binding of rigid H-NS filaments upstream and downstream of promoters, as is the Current Opinion in Microbiology 2012, 15:175–181
case at proV and bgl, occludes RNA polymerase or prevents the formation of an open complex, especially if the filament can span across the unbound region. MvaT, Lsr2, and the H-NS paralog StpA, each of which can functionally complement H-NS in vivo [63,64], have all been found to bridge DNA [48,54], and whether these molecules can also bind DNA in the stiffening mode has yet to be tested.
How does H-NS downregulate transcription? Remarkably another point of controversy is how H-NS binding functions to downregulate transcription. Several distinct mechanisms of H-NS silencing have been proposed at different promoters, which are conceivably consistent with either the stiffening or bridging mode of www.sciencedirect.com
Silencing of foreign DNA in bacteria Ali et al. 179
DNA binding. Some promoters sensitive to local topology may be locked in an inactive state due to the ability of H-NS to constrain supercoils [65]. H-NS binding at some promoters may occlude core promoter elements from binding by RNAP. At the rrnB promoter H-NS blocks transcription apparently by trapping it in an open complex but preventing elongation [66]. Two studies that tried to address whether trapping of RNAP by H-NS is the predominant mechanism of silencing in vivo using chromatin immunoprecipitation came to opposite conclusions, where one study suggested trapping is common at many promoters (based on H-NS/ RNAP co-occupancy) while the other study suggested H-NS occludes RNAP at most promoters [14,67]. It has been proposed that polymerase trapping is due to the formation of a repressory loop between H-NS linked regions [68,69]. Indeed a ‘trapped’ RNAP complex at the hdeAB promoter that involved DNA bridging by H-NS was visualized by atomic force microscopy [69]. In this case, RNAP in complex with the housekeeping sigma factor s70 (RNAPs70) facilitated the formation of H-NS bridges whereas RNAPs38 did not. If the stiffening model of DNA binding prevails these data will have to be revisited with more in depth mechanistic studies to determine why RNAPs38 is able to counter H-NS-mediated repression at promoters where RNAPs70 is blocked. Regardless, an understanding of the mechanism by which H-NS prevents transcription is critical to understanding how H-NS can be antagonized to activate transcription of foreign genes under physiologically appropriate conditions. Excellent reviews on diverse mechanisms of countersilencing have recently been written [5,11,28].
under physiologically appropriate conditions. The use of high-resolution visualization and single molecule biophysical approaches have yielded important insights but have also provided contradictory data. It is important that any hypothesis generated from biophysical data be congruent with genetic, high-throughput, and biochemical data generated by more traditional approaches.
Where to go from here
1.
Dobrindt U, Chowdary MG, Krumbholz G, Hacker J: Genome dynamics and its impact on evolution of Escherichia coli. Med Microbiol Immunol 2010, 199:145-154.
2.
Hacker J, Kaper JB: Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol 2000, 54:641-679.
3.
Ochman H, Lawrence JG, Groisman EA: Lateral gene transfer and the nature of bacterial innovation. Nature 2000, 405:299-304.
4.
Groisman EA, Ochman H: Pathogenicity islands: bacterial evolution in quantum leaps. Cell 1996, 87:791-794.
The recent advances in understanding the mechanism of how xenogeneic silencers bind DNA reveals that they target and repress foreign sequences based on their topological differences from the host chromosome. A recent study of chromatin structure in live cells suggesting that H-NS regulated genes may form distinct topological clusters distinct from the remainder of the chromosome, a finding that may also support bridging or other forms high-order association by H-NS in vivo [70]. We still, however, lack a clear mechanistic understanding of how H-NS binding effectively downregulates gene expression and how it assembles into a nucleoprotein structure that can be selectively antagonized under the appropriate conditions. We also lack any understanding of the role accessory molecules like Hha and YdgT play in silencing by H-NS [71,72]. The barriers to progress are due in part to the fact that H-NS has several unique properties that make it more challenging than ‘typical’ DNA binding proteins to study. Future progress will require the use of multiple complementary experimental approaches including well-controlled in vitro transcription assays and biophysical measurements www.sciencedirect.com
It is remarkable that Lsr2 and H-NS, which bear no similarity either in sequence or overall structure, and coming from species separated by over two billion years of evolution, have independently arrived at a common mechanism to target AT-rich DNA. Additional insights may still come through the comparative analysis of H-NS to functional analogs from other species. It is likely that the ability of silencers from different families to complement one another in vivo means their structures in the context of a nucleoprotein filament (either by bridging or stiffening) will be similar as well.
Acknowledgements The Navarre laboratory is supported by an Operating Grant and New Investigator Award from the Canada Institutes for Health Research (MOP86683 and MSH-87729) and a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN 386286-10). JL is supported by a CIHR Operating Grant (MOP-15107) and BX by Grant No. 2009CB521703 from the 973 Program of China. SSA is supported by a graduate fellowship from the Natural Sciences and Engineering Research Council of Canada.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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73. Sette M, Spurio R, Trotta E, Brandizi C, Brandi A, Pon CL, Barbato G, Boelens R, Gualerzi CO: Sequence-specific recognition of DNA by the C-terminal domain of nucleoidassociated protein H-NS. J Biol Chem 2009, 284:30453-30462. This study provides insight into the critical determinants of DNA binding by H-NS and why the TpA step is an important feature of high-affinity binding sites.
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