The Integration Host Factor is a pH-responsive protein that switches from DNA bending to DNA bridging in acidic biofilm-like conditions

TL;DR

Investigation of IHF-DNA interactions across a pH spectrum mimicking the acidic microenvironments of bacterial biofilms demonstrates that pH significantly modulates IHF-DNA interactions and explains the structural role played by IHF in supporting biofilm mechanics through intermolecular crosslinking.

Abstract

The Integration Host Factor (IHF) is a nucleoid-associated protein critical for both DNA compaction and biofilm stability. While its role in DNA packaging within the cell is well understood, its structural role in scaffolding biofilms is more puzzling and difficult to reconcile with its known DNA bending activity. Here, we investigated how IHF-DNA interactions are modulated across a pH spectrum mimicking the acidic microenvironments of bacterial biofilms. By performing all-atom calculations we discovered that low pHs lead to a change in protonation of IHF residues, which in turn exposes positively charged patches. We then conjectured that these positively charged residues could lead to intermolecular DNA bridging and tested this hypothesis through single-molecule and bulk assays. We discovered that while at physiological pH IHF mostly bends DNA, at pH < 5 there is clear evidence of IHF-mediated intermolecular crosslinking. Our results demonstrate that pH significantly modulates IHF-DNA interactions and explains the structural role played by IHF in supporting biofilm mechanics through intermolecular crosslinking.

Figures (5)

• Figure 1: Roles of IHF in cells and biofilms.a Within bacterial cells, IHF contributes to genome packaging by inducing sharp DNA bending Yoshua2021IHF. b In the biofilm, IHF contributes to strengthening the extracellular matrix novotny2013structural. We lack a quantitative understanding of the mechanisms underpinning the role of IHF in the mechanical stability of biofilms.
• Figure 2: All-atom simulations suggest pH-dependent protonation of IHF residues drives non-specific DNA binding.Top: Close-up view of all the amino acid residues that change protonation state as pH decreases. Each residue is labeled with the pH value at which its protonation state changes relative to neutral pH. At lower pH, acidic residues (Asp and Glu) shift from negatively charged to neutral, while histidine (His) transitions from neutral to protonated. Bottom: Comparison between the specific DNA–IHF complex (from PDB ID: 1IHF) and non-specific DNA bridging configurations obtained from all-atom MD simulations (this work). In all panels, DNA is shown in black, positively charged residues in blue, negatively charged in red, polar residues in pink, and apolar residues in gray.
• Figure 3: Effect of pH on IHF-mediated DNA compaction.a. AFM images of linearized pUC19 DNA at pH 7.5, 6.5, and 5.5 in the absence (top row) and presence (bottom row) of IHF. Scale bar corresponds to 100 nm b. Radius of gyration ($R_g$) across pH 7.5, 6.5, and 5.5, with Mann-Whitney U pairwise tests. $R_g$ distributions visualized as split violin plots (Left: Control/no IHF; Right: Sample with IHF). The mean $R_g$ was significantly smaller when IHF is present, and consistently small at lower pH for both, presence and absence, of IHF.
• Figure 4: DNA stretching reveals IHF-mediated DNA loops at low pH. a Sketch of the optical tweezer experiment at neutral and low pH. b Force-extension curves recorded for $\lambda$-DNA incubated with and without IHF at different pH conditions. Note the presence of sawtooth pattern at low pH in presence of IHF, suggesting crosslinks and DNA loops that are broken in the extension phase. c Persistence length $L_p$ of $\lambda$DNA determined by fitting the curves to the worm-like chain model across conditions, showing effective softening (shorter $L_p$) due to IHF binding. d Curves obtained by subtracting the retraction (or "unload") curves to the extension (or "load") curves. Values larger than zero suggest hysteresis, i.e. that extension requires more force that the retraction. We define the area under this curve $\Delta$ and it represent the dissipated energy during the extension/retraction cycle. e Dissipated energy $\Delta$ across conditions and showing that at low pH (5.5 and 4.5) the presence of IHF creates significant DNA loops that need breaking to extend DNA.
• Figure 5: Microrheology of IHF-DNA solutionsa, left Mean squared displacement of dense solutions of $\lambda$-DNA and IHF at neutral pH 7.5 and at different stoichiometries (DNA:protein ratio in legend). a, right Diffusion coefficient extracted from the long time behaviour of the MSDs. b, left Mean squared displacement of dense solutions of $\lambda$-DNA and IHF at neutral pH 4.5 and at different stoichiometries (DNA:protein ratio in legend). b, right Diffusion coefficient extracted from the long time behaviour of the MSDs. These results sugges that IHF is a "fluidifier" at neutral pH and a "thickener" at low pH.