How Proteins Remodel DNA in Bacteria under Stress

SCIENTIFIC ACHIEVEMENT

Multiscale, multimodal visualization techniques at the Advanced Light Source (ALS) enabled researchers to clarify how proteins remodel bacterial DNA in response to stressful environments.

SIGNIFICANCE AND IMPACT

The discovery could lead to new strategies for controlling microbial behavior and, eventually, new ways to fight bacterial infections.

In the center of the image is an animation of the interactions between DNA and HU proteins. On either side of that are animations of two rotating E. coli cells, with transparent cell walls. The cell on the left is labeled “neutral” and displays a compact yellow region inside. The cell on the right is labeled “acidic” and has a more diffuse yellow region. — Change in the architecture of the bacterial chromosome during the adaptation to an acidic environment is controlled by the DNA binding protein called HU (shown in shades of blue) and its interaction with DNA (yellow). (Credit: Michal Hammel/Berkeley Lab)

Bacterial rapid response

When bacteria are put in different environments, such as one that is more acidic or anaerobic, their genes start to adapt remarkably quickly. They’re able to do so because the proteins making up their chromosomes can pack and unpack rapidly, regulating the expression of genes that could help them respond to the changing environment. Now, researchers have visualized this process at the molecular level using advanced imaging techniques, an achievement that could eventually enable scientists to develop strategies to control microbial behavior.

DNA-packing proteins

Cartoon of four horizontal tubular shapes held in place by connecting protein structures. — HU dimers (blue) link up to form a network that arranges DNA strands (yellow) into an orderly parallel bundle.

In bacteria, the proteins responsible for DNA packing are called HU proteins. They are dimers—made up of two identical or nearly identical subunits that, when joined, form a bilaterally symmetrical “body” with two “arms.” To perform their packing function, these dimers “hug” the DNA, with arms on either side of a strand. They link up with each other (multimerize) to form arrays that hold the DNA strands parallel to each other. Changes that loosen or tighten this DNA bundling make the genetic information in the DNA more or less accessible to the enzymes responsible for gene expression.

In earlier work, the researchers used the ALS’s complementary visualization capabilities to show how HU proteins can trigger pathogenicity in E. coli. In this work, the researchers focused on how external environmental factors, such as acidity or salinity, affect HU function.

Visualization in three ways

For a multiscale perspective on DNA packing by HU under various conditions (different pH levels and salt concentrations, different growth phases, and wild type vs mutated), the researchers used three x-ray visualization techniques at the ALS. At the microscale, soft x-ray tomography at Beamline 2.1 enabled characterization of the higher-order organization of the E. coli nucleoid (the bacterial analogue of a cell nucleus). At the mesoscale, small-angle x-ray scattering (SAXS) at Beamline 12.3.1 made it possible to determine the overall shapes of HU–DNA complexes in solution. At the nanoscale, protein crystallography at Beamline 8.3.1 allowed identification of the molecular-level mechanisms that mediate DNA bundling.

The combination of SAXS and crystallography was crucial to gaining a detailed understanding of the HU response to environmental changes. Ready access to all three beamlines, as well as to experts onsite, greatly facilitated the work. Finally, the researchers also performed gene expression measurements, to correlate expression levels with the tomographic and scattering data.

Two graphs, each with four stacked SAXS curves. To the right of each graph, illustrations depict the corresponding HU–DNA configurations. — SAXS curves for wild-type (WT) HU–DNA complexes at two values of pH (4.5 and 5.5) and four salt (NaCl) concentrations (50 to 300 mM). Under most of these conditions, DNA strands (yellow) readily self-assemble into bundled structures, facilitated by HU proteins (blue). (a) At pH 4.5, the peak in the 1D SAXS profile corresponds to a DNA separation of 42 Å. (b) At pH 5.5, the peaks correspond to spacings of 70, 60, and 42 Å, depending on salt concentration. At both pH levels, the bundles separate into filament-like structures when the salt concentration reaches 300 mM.

Surviving the acid test

The tomography results revealed that, under acidic conditions, the chromatin is less condensed, with more diffuse borders. This “remodeling” of the chromatin was correlated with an increase in gene expression, consistent with the need to respond quickly to survive the stress of an acidic environment. The SAXS and crystallography data showed that HU multimerization is dependent on pH and salt concentrations. These effects mechanistically rely on HU’s promiscuity in forming multiple electrostatically driven multimerization interfaces. The next step will be to figure out how to control DNA packing in order to change bacterial behavior, with the ultimate goal of developing new approaches to fighting bacterial infections.

Orthogonal views of HU–DNA ribbon-diagram structures for pH 4.5 on left and pH 5.5 on right. — Crystal structures of HU–DNA complexes at pH 4.5 and 5.5, showing details of the molecular-level mechanism that alters the DNA bundling. Red and blue spheres indicate electrostatically charged residues involved in HU multimerization.

Contact: Michal Hammel

Researchers: S.G. Remesh (National Cancer Institute and Berkeley Lab); S.C. Verma and S. Adhya (National Cancer Institute); J.-H. Chen, A.A. Ekman, and C.A. Larabell (Berkeley Lab and Univ. of California, San Francisco); and M. Hammel (Berkeley Lab).

Funding: U.S. Department of Energy (DOE), Office of Science, Biological and Environmental Research program; National Institutes of Health; University of California Office of the President; Plexxicon Inc.; and International Union of Crystallography. Operation of the ALS is supported by the DOE Office of Science, Basic Energy Sciences program.

Publication: S.G. Remesh, S.C. Verma, J.-H. Chen, A.A. Ekman, C.A. Larabell, S. Adhya, and M. Hammel, “Nucleoid remodeling during environmental adaptation is regulated by HU-dependent DNA bundling,” Nat. Commun. 11, 2905 (2020), doi:10.1038/s41467-020-16724-5.

Adapted from the Berkeley Lab news release, “Study Gains New Insight Into Bacterial DNA Packing.”

ALS SCIENCE HIGHLIGHT #426