Bacterial histone HBb from Bdellovibrio bacteriovorus compacts DNA by bending

Abstract

Histones are essential for genome compaction and transcription regulation in eukaryotes, where they assemble into octamers to form the nucleosome core. In contrast, archaeal histones assemble into dimers that form hypernucleosomes upon DNA binding. Although histone homologs have been identified in bacteria recently, their DNA-binding characteristics remain largely unexplored. Our study reveals that the bacterial histone HBb (Bd0055) is indispensable for the survival of Bdellovibrio bacteriovorus, suggesting critical roles in DNA organization and gene regulation. By determining crystal structures of free and DNA-bound HBb, we unveil its distinctive dimeric assembly, diverging from those of eukaryotic and archaeal histones, while also elucidating how it binds and bends DNA through interaction interfaces reminiscent of eukaryotic and archaeal histones. Building on this, by employing various biophysical and biochemical approaches, we further substantiated the ability of HBb to bind and compact DNA by bending in a sequence-independent manner. Finally, using DNA affinity purification and sequencing, we reveal that HBb binds along the entire genomic DNA of B. bacteriovorus without sequence specificity. These distinct DNA-binding properties of bacterial histones, showcasing remarkable similarities yet significant differences from their archaeal and eukaryotic counterparts, highlight the diverse roles histones play in DNA organization across all domains of life.

Graphical Abstract

Figure 1.

Biophysical characterization of HBb. (A) Sequence alignment of representative eukaryotic, archaeal, and bacterial histones from Homo sapiens (UniProtKB/Swiss-Prot entry for H2A: P04908; H2B: P62807; H3: P68431; H4: P62805), Methanothermus fervidus (HMfA: P48781; HMfB: P19267), Bdellovibrio bacteriovorus (Q6MRM1), Leptospira interrogans (Q8F3E8), Plesiocystis pacifica (A6GF99), Enhygromyxa salina (NCBI entry WP_052547863.1), and Nannocystis exedens (WP_096326703.1). α-Helices are labeled with ‘h’ according to the crystal structures of H2A, HMfB and HBb, respectively. Conserved residues involved in DNA binding and tetramerization are highlighted in dark and light green, respectively. HBb residues involved in DNA backbone interaction according to the solved structures HBb-DNA_1 (PDB: 9EZZ) and HBb-DNA_2 (PDB: 9F0E) are in bold. Residues represented as sticks in Figure 3A and B are labeled with *. Residues R51 and D58 forming the RD clamp in HBb are connected by a bracket. (B) SDS-PAGE and BN-PAGE showing purified HBb. Triangles mark HBb on BN-PAGE. (C) Biophysical characterization of HBb by CD spectroscopy (left: single CD spectrum, middle: thermal melting curve) and SEC-MALS (right) for determination of secondary structure, thermal stability, and oligomeric state.

Figure 2.

HBb binds DNA. (A) EMSA with the 80 bp DNA fragment in the presence of HBb and HMfB as a control. The molar ratios of protein:DNA in lanes 4–10 are indicated. (B) MST curves showing binding of HBb to the 80 bp DNA fragment and the 80 bp-GC50 DNA fragment (10 nM), as compared to HMfB. (C) EMSA of HBb and HMfB as a control with the GeneRuler Ultra Low Range DNA Ladder (Thermo Fisher Scientific). For lanes 2–9, the protein/DNA mass ratios are labeled.

Figure 3.

Crystal structures of HBb in its free and DNA-bound forms. (A) Crystal structure of the HBb dimer (PDB: 8CMP) in cartoon representation. Selected residues are shown as sticks and conserved residues predicted to be involved in DNA binding or stabilization of the histone fold are in green. The salt bridges formed between D49, R51, and D58 are indicated as dashed lines. A representative 2Fo – Fc electron density map is shown for selected residues at a contour level of at 2.0 σ. (B) Crystal structure of the HMfB dimer (PDB: 1A7W) in cartoon representation for comparison. Conserved residues playing a role in DNA binding or stabilization of the histone fold are depicted as sticks. (C) Crystal structure of HBb-DNA_1 (PDB: 9EZZ) in cartoon representation with residues involved in DNA binding shown as sticks. The dashed frame marks the enlarged image section. (D) Crystal structure of HBb-DNA_2 (PDB: 9F0E) in cartoon representation with DNA- binding residues shown as sticks. The enlarged image section is marked by a dashed square. (E) Crystal packing of HBb-DNA_1 with selected symmetry mates generated within 4 Å. (F) Crystal packing of HBb-DNA_2 with selected symmetry mates generated within 20 Å. (G) Superposition of structures HBb-DNA_1, HBb-DNA_2, and their symmetrical mates, with DNA fragments trimmed for visualization. In all panels, the contents of a single asymmetric unit are shown in color and selected symmetry mates in gray. Hydrogen bonds and salt bridges are indicated as dashed lines.

Figure 4.

HBb is a DNA bender. (A) TPM experiment of HBb with 685 bp DNA. All measurement points are averages of triplicate measurements, with error bars indicating standard deviations. As reference, the RMS value obtained for hypernucleosome structure formed with HMfB is shown as the gray dash-dotted line (19). (B) MNase digestion of 685 bp DNA following incubation with HBb and HMfB. Increasing amounts of MNase in the assay are indicated in units. (C) DNA topology assay with relaxed pUC19 in the presence of HBb in comparison to HMfB. Protein:DNA mass ratios were labeled in the figure. The bands showing relaxed (△) and supercoiled pUC19 (▴) are marked. (D) Ligase-mediated circularization assay of a 240 bp DNA in the presence of HBb and HMfB. Samples are shown before and after T5 exonuclease digestion. Protein:DNA mass ratios are labeled. Monomeric DNA rings are indicated (*).

Figure 5.

MD simulations supporting DNA bending by HBb. (A) The HBb-DNA starting model used for MD simulation. The HBb-DNA model was constructed based on HBb-DNA crystal structures followed by energy minimization, and temperature and pressure equilibration. (B) Plots showing the bending angle of the DNA during two independent 500 ns simulations. (C) The enlarged image section of the DNA-bound HBb structure obtained from MD simulations. The structure shown is the central structure of the most common structure cluster in the last 100 ns of the simulations. Residues with a large number of contacts to DNA are shown as sticks. (D) The side view and top view of the cartoon representation of the central structure of the most common cluster of the simulations. (E) The side view and top view of a section of HMfB bound to DNA (PDB: 5T5K). The cartoon representation shows an HMfB dimer with a 38-bp dsDNA wrapping around it.

Figure 6.

The HBb dimer binds DNA nonspecifically. (A) DAP-seq data visualized using a circos plot of the B. bacteriovorus HD100 genome. The inner rug plots represent the ‘minus’ (yellow) and ‘plus’ (light green) strand ORFs. Each wedge represents the coordinates of a gene. Outer line plots represent the normalized read coverage values, and scatter plots above show the major peaks for the two DAP-seq samples (blue and purple). Peaks are shown as dots, where the strength of the peak signal correlates positively with the distance from the axis. The enlarged section shown in panel E is indicated by gray dashed line. (B) Line plots showing the normalized read coverage values for a randomly selected section of the B. bacteriovorus HD100 genome encompassing the genomic region from 151 464 bp to 189 033 bp. The bottom plot shows the genomic context, and the top line plots depict the normalized read coverage values for each DAP-seq sample with the major peaks shaded darker. The color code corresponds to panel A.