Fis Binding Sites Research Articles

The DNA relaxation-dependent OFF-to-ON biasing of the type 1 fimbrial genetic switch requires the Fis nucleoid-associated protein.

The structural genes expressing type 1 fimbriae in Escherichia coli alternate between expressed (phase ON) and non-expressed (phase OFF) states due to inversion of the 314 bp fimS genetic switch. The FimB tyrosine integrase inverts fimS by site-specific recombination, alternately connecting and disconnecting the fim operon, encoding the fimbrial subunit protein and its associated secretion and adhesin factors, to and from its transcriptional promoter within fimS. Site-specific recombination by the FimB recombinase becomes biased towards phase ON as DNA supercoiling is relaxed, a condition that occurs when bacteria approach the stationary phase of the growth cycle. This effect can be mimicked in exponential phase cultures by inhibiting the negative DNA supercoiling activity of DNA gyrase. We report that this bias towards phase ON depends on the presence of the Fis nucleoid-associated protein. We mapped the Fis binding to a site within the invertible fimS switch by DNase I footprinting. Disruption of this binding site by base substitution mutagenesis abolishes both Fis binding and the ability of the mutated switch to sustain its phase ON bias when DNA is relaxed, even in bacteria that produce the Fis protein. In addition, the Fis binding site overlaps one of the sites used by the Lrp protein, a known directionality determinant of fimS inversion that also contributes to phase ON bias. The Fis–Lrp relationship at fimS is reminiscent of that between Fis and Xis when promoting DNA relaxation-dependent excision of bacteriophage λ from the E. coli chromosome. However, unlike the co-binding mechanism used by Fis and Xis at λ attR, the Fis–Lrp relationship at fimS involves competitive binding. We discuss these findings in the context of the link between fimS inversion biasing and the physiological state of the bacterium.

Unraveling the Complex Interplay of Fis and IHF Through Synthetic Promoter Engineering.

Bacterial promoters are usually formed by multiple cis-regulatory elements recognized by a plethora of transcriptional factors (TFs). From those, global regulators are key elements since these TFs are responsible for the regulation of hundreds of genes in the bacterial genome. For instance, Fis and IHF are global regulators that play a major role in gene expression control in Escherichia coli, and usually, multiple cis-regulatory elements for these proteins are present at target promoters. Here, we investigated the relationship between the architecture of the cis-regulatory elements for Fis and IHF in E. coli. For this, we analyze 42 synthetic promoter variants harboring consensus cis-elements for Fis and IHF at different distances from the core −35/−10 region and in various numbers and combinations. We first demonstrated that although Fis preferentially recognizes its consensus cis-element, it can also recognize, to some extent, the consensus-binding site for IHF, and the same was true for IHF, which was also able to recognize Fis binding sites. However, changing the arrangement of the cis-elements (i.e., the position or number of sites) can completely abolish the non-specific binding of both TFs. More remarkably, we demonstrated that combining cis-elements for both TFs could result in Fis and IHF repressed or activated promoters depending on the final architecture of the promoters in an unpredictable way. Taken together, the data presented here demonstrate how small changes in the architecture of bacterial promoters could result in drastic changes in the final regulatory logic of the system, with important implications for the understanding of natural complex promoters in bacteria and their engineering for novel applications.

Colonization efficiency of Pseudomonas putida is influenced by Fis-controlled transcription of nuoA-N operon.

Root colonization of plant growth-promoting bacteria is a complex multistep process that is influenced by several factors. For example, during adherence to plant roots, bacteria have to endure reactive oxygen species (ROS) produced by plants. In this study, we report that the global transcriptional regulator Fis is involved in the regulation of ROS-tolerance of Pseudomonas putida and thereby affects barley root colonization. Fis overexpression reduced both ROS-tolerance and adherence to barley roots and activated the transcription of the nuoA-N operon encoding NADH dehydrogenase I, the first enzyme of a membrane-bound electron-transport chain. The nuoA-N knockout mutation in the fis-overexpression background increased the ROS-tolerance and adherence to barley roots. We show that nuoA has two transcriptional start sites located 104 and 377 nucleotides upstream of the coding sequence, indicating the presence of two promoters. The DNase I footprint analysis revealed four Fis binding sites: Fis-nuo1 to Fis-nuo4, situated between these two promoters. Site-directed mutagenesis in a promoter-lacZ reporter and β-galactosidase assay further confirmed direct binding of Fis to Fis-nuo2 and probably to Fis-nuo4 but not to Fis-nuo1 and Fis-nuo3. Additionally, the results implied that Fis binding to Fis-nuo4 could affect transcription of the nuoA-N operon by modification of upstream DNA topology. Moreover, our transposon mutagenesis results indicated that Fis might be involved in the regulation of several alternative ROS detoxification processes utilizing NADH.

Physical properties of DNA may direct the binding of nucleoid-associated proteins along the E. coli genome

Nucleoid-associated proteins (NAPs) play important roles in both chromosome packaging and gene regulation in bacteria. The underlying mechanisms, however, remain elusive particularly for how NAPs contribute to chromosome packaging. We report here a characterization of the binding sites for several major NAPs in E. coli, namely HNS, IHF, Fis, Dps and a non-NAP protein, FNR, in terms of the physical properties of their binding DNA. Our study shows that (i) as compared with flanking regions, the binding sites for IHF, Fis and FNR tend to have high intrinsic curvature, while no characterized pattern of intrinsic curvature distribution around those of HNS and Dps; (ii) all the binding sites analyzed in this study except those of HNS are characterized by high structural flexibility; (iii) the intrinsic curvature and flexibility at the binding sites for Fis and IHF are found to be coupled with the sequence specificity required in their binding, while the physical properties of the binding regions for both Dps and FNR are independent of sequence specificity. Our data suggest that physical properties of DNA sequence may contribute to binding of NAPs and mediate genome packaging and transcriptional regulation of the downstream genes. Our results should be informative for prediction of NAPs binding sites and understanding of the bacterial chromosome packaging.

The promoter region of lapA and its transcriptional regulation by Fis in Pseudomonas putida.

LapA is the biggest protein in Pseudomonas putida and a key factor for biofilm formation. Its importance and posttranslational regulation is rather thoroughly studied but less is known about the transcriptional regulation. Here we give evidence that transcription of lapA in LB-grown bacteria is initiated from six promoters, three of which display moderate RpoS-dependence. The global transcription regulator Fis binds to the lapA promoter area at six positions in vitro, and Fis activates the transcription of lapA while overexpressed in cells. Two of the six Fis binding sites, Fis-A7 and Fis-A5, are necessary for the positive effect of Fis on the transcription of lapA in vivo. Our results indicate that Fis binding to the Fis-A7 site increases the level of transcription from the most distal promoter of lapA, whereas Fis binding to the Fis-A5 site could be important for modifying the promoter area topology.

Temporal control of Dickeya dadantii main virulence gene expression by growth phase-dependent alteration of regulatory nucleoprotein complexes

In bacteria, important genes are often controlled at the transcriptional level by several factors, forming a complex and intertwined web of interactions. Yet, transcriptional regulators are often studied separately and little information is available concerning their interactions. In this work, we dissect the regulation of the major virulence gene pelD in D. dadantii by taking into account the effects of individual binding sites for regulatory proteins FIS and CRP, and the impact of a newly discovered divergent promoter div. Using a combination of biochemistry and genetics approaches we provide an unprecedented level of detail on the multifactorial regulation of bacterial transcription. We show that the growth phase dependent regulation of pelD is under the control of changing composition of higher-order nucleoprotein complexes between FIS, CRP, div and pelD during the growth cycle that allow sequential expression of div and pelD in the early and late exponential growth phases, respectively. This work highlights the importance of “orphan” promoters in gene regulation and that the individual binding sites for a regulator can serve several purposes and have different effects on transcription, adding a new level of complexity to bacterial transcriptional regulation.

DNA Sequence Determinants Controlling Affinity, Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis.

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.

Effect of FIS on Transcription-Coupled DNA Supercoiling in E. coli

For decades, it has been recognized that transcribing along the DNA double helix by a RNA polymerase can enhance localized DNA supercoiling. This process has been elegantly explained by a “twin-supercoiled-domain” model of transcription in which positive DNA supercoils are generated ahead of a translocating RNA polymerase and negative supercoils behind it. In this study, we utilized bacterial genetics and biochemical approaches to investigate how FIS protein regulates transcription-coupled DNA supercoiling (TCDS) by the strong rrnB P1, P2 promoters. We first generated several E. coli strains including MG1655(DE3)ΔfisΔlacZ and VS111(DE3)ΔfisΔlacZ using the λ Red recombination system. We then inserted the divergently coupled Pleu-500 and IPTG-inducible rrnB P1, P2 promoters with the luc and lacZ genes to the attTn7 site of the E. coli chromosome. Additionally, the upstream region of the rrnB P1 promoter including the three FIS-binding sites was placed between the Pleu-500 and the rrnB P1, P2 promoters. Our results showed that TCDS initiated from the rrnB P1, P2 promoters greatly activated the divergently coupled Pleu-500 promoter. Intriguingly, we found that FIS protein activated the transcription from the P1 and P2 promoters and potently inhibited the activation of the Pleu-500 promoter by TCDS. Our results are consistent with a model in which FIS protein forms topological barriers upon binding to its recognition sites in the upstream region of the rrnB P1 promoter, blocks TCDS diffusion, and potently inhibits the activation of the Pleu-500 promoter by TCDS. Likewise, TCDS generated from the strong P1 and P2 promoters bounced back and activated the P1 and P2 promoters. We also used atomic force microscopy to examine the shape and volume of these E. coli mutant strains.

Facilitated Dissociation of Protein from a Single DNA Binding Site

Protein-DNA interactions are central to chromosome compaction, defining the topology of chromatin, DNA repair, and gene regulation, and therefore control all aspects of cellular function. It is therefore important to understand the factors that regulate the dynamics of protein-DNA interactions since these factors will influence the dynamics of cellular processes. Previous single DNA studies involving the major nucleoid-associated protein Fis have shown that it stably binds 48.5 kb DNA in Fis-free buffer. However, protein in solution was found to accelerate the off-rate of Fis in a concentration dependent manner via an unknown mechanism. This effect has also recently been observed in a number of other cases suggesting its generality. It is unknown whether facilitated dissociation is an effect at the single binding site level or whether it involves protein clustering and/or cooperativity. Using single molecule fluorescence microscopy, we have measured the off-rate of Fis from 27bp dsDNAs, constituting individual Fis binding sites, and have observed facilitated exchange demonstrating an effect at the single binding site level. We have also found that the salt dependence of the off-rate is dramatically reduced when protein is in solution. This observation provides strong support for a simple microscopic theory that thermally excited, partial dissociation events lead to facilitated dissociation. In the absence of cooperativity, it is commonly assumed that finding the ratio of the off-rate to the on-rate constant will produce the same dissociation constant as finding the concentration of ligand for which half of the substrates are bound by ligand. However, if the off-rate is a function of protein concentration, this equivalence no longer holds. If facilitated exchange is found to be a general phenomenon, it will force a change in how we think about protein binding kinetics in biology.

Pseudomonas putida Fis binds to the lapF promoter in vitro and represses the expression of LapF.

The biofilm matrix of the rhizospheric bacterium Pseudomonas putida consists mainly of a proteinaceous component. The two largest P. putida proteins, adhesins LapA and LapF, are involved in biofilm development but prevail in different developmental stages of the biofilm matrix. LapA is abundant in the initial stage of biofilm formation whereas LapF is found in the mature biofilm. Although the transcriptional regulation of the adhesins is not exhaustively studied, some factors that can be involved in their regulation have been described. For example, RpoS, the major stress response sigma factor, activates, and Fis represses LapF expression. This study focused on the LapF expression control by Fis. Indeed, using DNase I footprint analysis a Fis binding site Fis-F2 was located 150 bp upstream of the lapF gene coding sequence. The mapped 5′ end of the lapF mRNA localized the promoter to the same region, overlapping with the Fis binding site Fis-F2. Monitoring the lapF promoter activity by a β-galactosidase assay revealed that Fis overexpression causes a 4-fold decrease in the transcriptional activity. Furthermore, mutations that diminished Fis binding to the Fis-F2 site abolished the repression of the lapF promoter. Thus, these data suggest that Fis is involved in the biofilm regulation via repression of LapF expression.

Binding of Nucleoid-Associated Protein Fis to DNA Is Regulated by DNA Breathing Dynamics

Physicochemical properties of DNA, such as shape, affect protein-DNA recognition. However, the properties of DNA that are most relevant for predicting the binding sites of particular transcription factors (TFs) or classes of TFs have yet to be fully understood. Here, using a model that accurately captures the melting behavior and breathing dynamics (spontaneous local openings of the double helix) of double-stranded DNA, we simulated the dynamics of known binding sites of the TF and nucleoid-associated protein Fis in Escherichia coli. Our study involves simulations of breathing dynamics, analysis of large published in vitro and genomic datasets, and targeted experimental tests of our predictions. Our simulation results and available in vitro binding data indicate a strong correlation between DNA breathing dynamics and Fis binding. Indeed, we can define an average DNA breathing profile that is characteristic of Fis binding sites. This profile is significantly enriched among the identified in vivo E. coli Fis binding sites. To test our understanding of how Fis binding is influenced by DNA breathing dynamics, we designed base-pair substitutions, mismatch, and methylation modifications of DNA regions that are known to interact (or not interact) with Fis. The goal in each case was to make the local DNA breathing dynamics either closer to or farther from the breathing profile characteristic of a strong Fis binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by gel-shift assay, changed in accordance with our expectations. We conclude that Fis binding is associated with DNA breathing dynamics, which in turn may be regulated by various nucleotide modifications.

Observation of Synapse Rotation of Engineered Recombinase HIN-H107Y

Site-specific recombination promotes integration, excision or inversion of DNA strands during gene transcription and chromosome replication. The recombination process generally involves the formation of synaptic complexes, DNA cleavage and rejoining, though the specific mechanism of each enzyme varies. The Hin recombinase is a member of the serine recombinase family which has an active serine residue that can catalyze the DNA cleavage and re-ligating. Hin functions to invert a 900 bp DNA segment between two hin-binding (hix) sites within the Salmonella chromosome that contains a promoter for downstream flagellar genes. The inversion requires the auxiliary protein Fis and two Fis-binding sites on the targeted DNA segment, which assemble together with hix sites and Hin to form an invertasome structure. The assembly is stimulated by the protein HU. A class of engineered Hin mutants Hin-H107Y (H107Y) is able to assemble oligonucleotide substrates containing hix sites into stable synaptic complexes that catalyze recombination without Fis and HU. Structural data suggest that Hin share with gamma-delta resolvase a similar helix-turn-helix domain at the 40-50 amino acid residues. We hypothesized that H107Y could act like resolvase to form four subunits synapse, cleave both strands of DNA within the center of hix site, rotate and rejoin. We therefore used single DNA assays to analyze the dynamics associated with H107Y catalyzed strand exchange.Enzyme-DNA binding and synapse formation was observed with a single DNA containing two hix sites. We further obtained data for subunits rotation and re-ligation from a braiding assay which consisted of double DNAs each having a single hix site, indicating that the rotation strand exchange mechanism observed recently for Bxb1 Int is also possessed by Hin.

76 Protein–DNA binding and breathing dynamics of DNA

In the living cell, the double-stranded DNA molecule experiences thermal motions that induce spontaneous openings and re-closings of the double helix known as “DNA breathing,” or “DNA transient bubbles” (Englander et al., 1980). The propensity for breathing is interconnected with DNA local stability and flexibility (Vafabakhsh & Ha, 2012), which play a key role in DNA biological function (Bishop et al., 2012). Here, we investigate the relationship between the DNA local propensity for breathing and binding of two transcription factors (TFs): (i) the human TF YY1, (see e.g. Usheva & Shenk, 1996), and (ii) the nucleoid-associated protein Fis in Escherichia coli (see e.g. Finkel & Johnson, 1992). Using a mesoscopic nonlinear model of double-stranded DNA (Peyrard & Bishop, 1989) that can be augmented for rational design of DNA breathing (Alexandrov et al., 2010), we have simulated the dynamics of known Fis- and YY1-binding sites, analyzed published in vitro and genomic data-sets, and conducted targeted experimental tests of our predictions (Alexandrov et al., 2012; Nowak-Lovato et al., in press). We found a strong correlation between the propensity for breathing (at the binding sites) and YY1/Fis binding. We identified a breathing profile that is characteristic for a strong Fis-binding site that is significantly enriched among the identified in vivo E. coli Fis-binding sites. To test our understanding of how Fis binding is influenced by the breathing, we designed base-pair substitutions, mismatch, and O6-Guanine methylation modifications of nucleotides, in sequences that are known to interact (or not interact) with Fis, seeking to make the breathing either closer to or farther from the breathing profile of a strong Fis-binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by EMSA, changed in accordance with our expectations. Further, by using site-specific chromatin immunopecipitations, BIOBASE data, and simulations, we also found a specific breathing profile at the binding cites of YY1 in vivo. Our finding suggests that the genomic-flanking sequence variations and SNPs presence may exert long-range effects on DNA breathing and predetermine YY1 binding in cells.

Altered regulation of the OmpF porin by Fis in Escherichia coli during an evolution experiment and between B and K-12 strains.

The phenotypic plasticity of global regulatory networks provides bacteria with rapid acclimation to a wide range of environmental conditions, while genetic changes in those networks provide additional flexibility as bacteria evolve across long time scales. We previously identified mutations in the global regulator-encoding gene fis that enhanced organismal fitness during a long-term evolution experiment with Escherichia coli. To gain insight into the effects of these mutations, we produced two-dimensional protein gels with strains carrying different fis alleles, including a beneficial evolved allele and one with an in-frame deletion. We found that Fis controls the expression of the major porin-encoding gene ompF in the E. coli B-derived ancestral strain used in the evolution experiment, a relationship that has not been described before. We further showed that this regulatory connection evolved over two different time scales, perhaps explaining why it was not observed before. On the longer time scale, we showed that this regulation of ompF by Fis is absent from the more widely studied K-12 strain and thus is specific to the B strain. On a shorter time scale, this regulatory linkage was lost during 20,000 generations of experimental evolution of the B strain. Finally, we mapped the Fis binding sites in the ompF regulatory region, and we present a hypothetical model of ompF expression that includes its other known regulators.

Role of Decreased Levels of Fis Histone-Like Protein in Crohn's Disease-Associated Adherent Invasive E scherichia coli LF82 Bacteria Interacting with Intestinal Epithelial Cells

The interaction of Crohn's disease (CD)-associated adherent-invasive Escherichia coli (AIEC) strain LF82 with intestinal epithelial cells depends on surface appendages, such as type 1 pili and flagella. Histone-like proteins operate as global regulators to control the expression of these virulence factors. We evaluated the role of histone-like proteins in AIEC reference strain LF82 during infection of intestinal epithelial cells, Intestine-407, and observed that the fis mRNA level was decreased. The role of Fis in AIEC LF82 was determined by studying the phenotype of an LF82 fis::Km mutant. This was the first mutant of strain LF82 that has been described thus far that is unable to express flagellin but still able to produce type 1 pili. The cyclic-di-GMP pathway linking flagella and type 1 pilus expression is not involved in Fis-mediated regulation, and we identified in the present study Fis-binding sites located upstream of the fimE gene and in the intergenic region between fimB and nanC of the fim operon encoding type 1 pili. The major consequence of decreased Fis expression in AIEC bacteria in contact with host cells is a direct downregulation of fimE expression, leading to the preferential ON phase of the fimS element. Thus, by maintaining type 1 pilus expression, AIEC bacteria, which interact with the gut mucosa, have greater ability to colonize and to induce inflammation in CD patients.

Competition between NarL-dependent activation and Fis-dependent repression controls expression from the Escherichia coli yeaR and ogt promoters

The Escherichia coli NarL protein is a global gene regulatory factor that activates transcription at many target promoters in response to nitrate and nitrite ions. Although most NarL-dependent promoters are also co-dependent on a second transcription factor, FNR protein, two targets, the yeaR and ogt promoters, are activated by NarL alone with no involvement of FNR. Biochemical and genetic studies presented here show that activation of the yeaR promoter is dependent on the binding of NarL to a single target centred at position -43.5, whereas activation at the ogt promoter requires NarL binding to tandem DNA targets centred at position -45.5 and -78.5. NarL-dependent activation at both the yeaR and ogt promoters is decreased in rich medium and this depends on Fis, a nucleoid-associated protein. DNase I footprinting studies identified Fis-binding sites that overlap the yeaR promoter NarL site at position -43.5, and the ogt promoter NarL site at position -78.5, and suggest that Fis represses both promoters by displacing NarL. The ogt gene encodes an O6-alkylguanine DNA alkyltransferase and, hence, this is the first report of expression of a DNA repair function being controlled by nitrate ions.

Loss of Hda activity stimulates replication initiation from I‐box, but not R4 mutant origins in Escherichia coli

Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA(ATP) specific sites (I-boxes, tau-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC. Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA(ATP) containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA(ATP) specific I-boxes. However, competition between I-box mutant and wild-type origins, revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA(ATP)/DnaA(ADP) ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.

Regulation by Nucleoid-Associated Proteins at the Escherichia coli nir Operon Promoter

The Escherichia coli K-12 nir operon promoter can be fully activated by binding of the regulator of fumarate and nitrate reduction (FNR) to a site centered at position -41.5 upstream of the transcript start, and this activation is modulated by upstream binding of the integration host factor (IHF) and Fis (factor for inversion stimulation) proteins. Thus, transcription initiation is repressed by the binding of IHF and Fis to sites centered at position -88 (IHF I) and position -142 (Fis I) and activated by IHF binding to a site at position -115 (IHF II). Here, we have exploited mutational analysis and biochemistry to investigate the actions of IHF and Fis at these sites. We show that the effects of IHF and Fis are position dependent and that IHF II functions independently of IHF I and Fis I. Using in vitro assays, we report that IHF and Fis repress transcription initiation by interfering with RNA polymerase binding. Differences in the upstream IHF and Fis binding sites at the nir promoter in related enteric bacteria fix the level of nir operon expression under anaerobic growth conditions.

Genome-wide analysis of Fis binding in Escherichia coli indicates a causative role for A-/AT-tracts

We determined the genome-wide distribution of the nucleoid-associated protein Fis in Escherichia coli using chromatin immunoprecipitation coupled with high-resolution whole genome-tiling microarrays. We identified 894 Fis-associated regions across the E. coli genome. A significant number of these binding sites were found within open reading frames (33%) and between divergently transcribed transcripts (5%). Analysis indicates that A-tracts and AT-tracts are an important signal for preferred Fis-binding sites, and that A(6)-tracts in particular constitute a high-affinity signal that dictates Fis phasing in stretches of DNA containing multiple and variably spaced A-tracts and AT-tracts. Furthermore, we find evidence for an average of two Fis-binding regions per supercoiling domain in the chromosome of exponentially growing cells. Transcriptome analysis shows that approximately 21% of genes are affected by the deletion of fis; however, the changes in magnitude are small. To address the differential Fis bindings under growth environment perturbation, ChIP-chip analysis was performed using cells grown under aerobic and anaerobic growth conditions. Interestingly, the Fis-binding regions are almost identical in aerobic and anaerobic growth conditions-indicating that the E. coli genome topology mediated by Fis is superficially identical in the two conditions. These novel results provide new insight into how Fis modulates DNA topology at a genome scale and thus advance our understanding of the architectural bases of the E. coli nucleoid.

Functional Characterization of the Escherichia coli Fis–DNA Binding Sequence

The Escherichia coli protein Fis is remarkable for its ability to interact specifically with DNA sites of highly variable sequences. The mechanism of this sequence-flexible DNA recognition is not well understood. In a previous study, we examined the contributions of Fis residues to high-affinity binding at different DNA sequences using alanine-scanning mutagenesis and identified several key residues for Fis–DNA recognition. In this work, we investigated the contributions of the 15-bp core Fis binding sequence and its flanking regions to Fis–DNA interactions. Systematic base-pair replacements made in both half sites of a palindromic Fis binding sequence were examined for their effects on the relative Fis binding affinity. Missing contact assays were also used to examine the effects of base removal within the core binding site and its flanking regions on the Fis–DNA binding affinity. The results revealed that: (1) the − 7G and + 3Y bases in both DNA strands (relative to the central position of the core binding site) are major determinants for high-affinity binding; (2) the C 5 methyl group of thymine, when present at the + 4 position, strongly hinders Fis binding; and (3) AT-rich sequences in the central and flanking DNA regions facilitate Fis–DNA interactions by altering the DNA structure and by increasing the local DNA flexibility. We infer that the degeneracy of specific Fis binding sites results from the numerous base-pair combinations that are possible at noncritical DNA positions (from − 6 to − 4, from − 2 to + 2, and from + 4 to + 6), with only moderate penalties on the binding affinity, the roughly similar contributions of − 3A or G and + 3T or C to the binding affinity, and the minimal requirement of three of the four critical base pairs to achieve considerably high binding affinities.