![]() ![]() We show that this domain drives ComEA oligomerization, and that oligomerization is required for genetic transformation. These studies reveal that the DNA binding domain is an atypical helix-hairpin-helix domain, and, most importantly, that a previously unidentified domain lies within the Gram-positive ComEA region of unknown function. subtilis (ComEA Bs) and Geobacillus stearothermophilus (ComEA Gs) together with complementary genetic and biophysical analyses. Here we present X-ray crystal structures of ComEA from B. To investigate this complex structure and to gain insight into its mode of action, we conducted an in vitro and in vivo structure-function investigation, centered on the B. The complex structure and membrane anchoring of ComEA in the Gram-positives suggests that it may act differently than the simple Brownian ratchet mechanism proposed for V. Equivalent Gram-negative proteins have not been identified. In Gram-positives, the membrane-associated ATPase ComFA and its partner protein ComFC, appear to provide the energy for transport. In all bacteria, following uptake to the periplasm, one strand of tDNA is degraded and the remaining strand is transported to the cytoplasm through the ComEC membrane channel (reviewed in 1, 2). ![]() Thus, the pilus initiates uptake while ComEA operates as a Brownian ratchet 12, providing the driving force for uptake of the bulk of the tDNA into the periplasm. It has been proposed that in the Gram-negative Vibrio cholerae and Neisseria gonorrhoeae, the single-domain ComEA then binds to this introduced tDNA segment, preventing loss of tDNA by backward diffusion across the outer membrane 6, 11. In both Gram-negative and positive bacteria, except for Helicobacter pylori 8, uptake is initiated by type 4 pili that bind DNA and retract, pulling a segment of transforming DNA into the periplasmic compartment 9, 10. In sharp contrast, ComEA in the Gram-negatives contains only a single (stand-alone) DNA-binding domain which is free to diffuse in the periplasm 5, 6, 7 (Fig. This membrane anchor and a C-terminal DNA binding domain are separated by a region of unknown function consisting of ~110 amino acids (Fig. subtilis and other Gram-positive bacteria, ComEA is bound to the membrane by an N-terminal transmembrane region 4. It was subsequently shown to be a non-specific DNA-binding protein required for both stable binding of tDNA to the cell and for tDNA uptake 3, 4. ComEA, was discovered in genetic screens for transformation deficiency in Bacillus subtilis. Following this contact with the tpilus, the movement of tDNA into the periplasm is driven by its interaction with the widely conserved periplasmic protein ComEA (Fig. Uptake is initiated at the cell surface when tDNA interacts with a transformation pilus (tpilus). Elements of the figure were created with. Residue numbering corresponds to ComEA Bs or ComEA Vc. The magenta line denotes a region of unknown function, which is addressed in this study. Residues 1–24 are not shown in order to highlight the fact that they comprise a predicted secretion signal that is cleaved to generate mature ComEA Vc, which diffuses freely in the periplasm. C Domain architecture of ComEA from a representative Gram-negative bacterium, V. B Domain architecture of ComEA from a representative Gram-positive bacterium, B. ComEC is proposed to degrade one strand of DNA and provide the channel for transport of DNA into the cytoplasm. Here, the DNA encounters ComEA, which stabilizes binding to the cell and propels continued uptake of the DNA. The tpilus binds weakly to DNA and retracts to pull it into the periplasm. The tpilus is believed to be anchored to the membrane protein ComGB. Similar content being viewed by othersĪ The tpilus is composed of ComGC, whose assembly requires the ATPase ComGA. 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. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. 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. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. 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.
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