Structure-function studies reveal ComEA contains an oligomerization domain essential for transformation in gram-positive bacteria

Abstract

An essential step in bacterial transformation is the uptake of DNA into the periplasm, across the thick peptidoglycan cell wall of Gram-positive bacteria, or the outer membrane and thin peptidoglycan layer of Gram-negative bacteria. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. Here we determine X-ray crystal structures of ComEA from two Gram-positive species, Bacillus subtilis and Geobacillus stearothermophilus, identifying a domain that is absent in Gram-negative bacteria. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. Finally, we present a model for the interaction of the ComEA DNA-binding domain with DNA, suggesting that ComEA oligomerization may provide a pulling force that drives DNA uptake across the thick cell walls of Gram-positive bacteria.

Chemically Tailored Single Atoms for Targeted and Light‐Controlled Bactericidal Activity

“…Conversely, gram‐positive bacteria possess a dense peptidoglycan layer that poses challenges for targeted binding. [ 24 ] Importantly, it is noteworthy that even under the highest inhibitory circumstances, the overall temperature of the bacterial suspension remains approximately 45 °C (Figure S11, Supporting Information), which is usually insufficient to kill planktonic bacteria. This suggests that the observed remarkable bactericidal impact probably attributed from the targeted binding, which enhances heat density on the bacterial surface and subsequently compromises the integrity of the cell membrane.…”

Methods for the Design and Analysis of Analytical Ultracentrifugation Experiments

“…MW‐AUC facilitates the characterization of hetero‐interactions when individual molecules with unique spectral chromophores can be separated in a MW‐AUC analysis. In such systems, it is even possible to determine the molar stoichiometry of assembly (Ahmed et al., 2022; Gabir et al., 2023; Henrickson et al., 2022; Horne et al., 2021; Horne et al., 2020; Johnson et al., 2018; Zhang et al., 2017). By analyzing protein molecules in a physiological solution environment, this technique provides valuable details about dynamic interactions, solution composition, conformation, and oligomerization properties of proteins.…”

Visualizing dynamic competence pili and DNA capture throughout the long axis ofBacillus subtilis

“…Once extended, the ComGC pilus binds to free dsDNA in the environment (20,21). Dynamic retraction of the ComGC subunits back into the membrane shortens the pilus, transporting bound DNA through the gap formed in the cell wall, into contact with the membrane-bound DNA receptor/binding protein ComEA (23)(24)(25)(26). After ComEA binding, DNA is brought into contact with the ComEC membrane channel (27,28).…”