Bakterieller Gen-Transfer - Eine Untersuchung auf Einzelmolekülniveau
Zusammenfassung der Projektergebnisse
In this project, we characterized DNA uptake during bacterial transformation at the single molecule level. At the start of the project, we had shown that the gram-positive Bacillus subtilis uses a molecular motor to take up DNA against external forces exceeding 50 pN. In the project, we mainly investigated the characteristics and molecular mechanism of motors driving DNA uptake through the outer membrane of gram-negative species. Neisseria gonorrhoeae, like almost all naturally competent species, uses its type 4 pilus system for DNA uptake during transformation, yet the molecular mechanism of uptake was unclear. In this project, we showed that DNA is imported into the periplasm at random locations around the cell contour. We found using fluorescent DNA that the periplasm is saturable within minutes with ~ 40 kbp DNA. The periplasmic DNA-binding protein ComE quantitatively governs the carrying capacity of the periplasm in a gene-dosage-dependent fashion. In the absence of external DNA, ComE is homogeneously distributed in the periplasm. Upon addition of external DNA, ComE is re-localized to form discrete foci colocalized with imported DNA. This suggested that ComE might act as a chaperone in a translocation ratchet and powers DNA uptake through the outer membrane. To assess this hypothesis, we used single-molecule techniques for characterizing the force-dependent velocity of DNA uptake. We found that the DNA uptake velocity depends on the concentration of ComE, indicating that ComE is directly involved in the uptake process. The velocity–force relation of DNA uptake is in very good agreement with a translocation ratchet model where binding of chaperones in the periplasm biases DNA diffusion through a membrane pore in the direction of uptake. The model yields a speed of DNA uptake of 900 bp·s^−1 and a reversal force of 17 pN. Moreover, by comparing the velocity–force relation of DNA uptake and type 4 pilus retraction, we can exclude pilus retraction as a mechanism for DNA uptake. In conclusion, our data strongly support the model of a translocation ratchet with ComE acting as a ratcheting chaperone. Helicobacter pylori is naturally competent, yet it uses a type 4 secretion system for DNA uptake. To find out whether the characteristics of DNA uptake are different in this species, we used our single molecule assays. Again, we localized imported fluorescent DNA in the periplasm. Single molecule analysis revealed that outer membrane DNA transport occurred at a velocity of 1.3 kbps^−1 and that previously imported DNA was reversibly extracted from the bacterium at pulling forces exceeding 23 pN. dsDNA was transiently detected in the periplasm in wild type H. pylori, but was periplasmatically trapped in a mutant lacking the B. subtilis membrane-channel homolog ComEC. We conclude that H. pylori uses a two-step DNA uptake mechanism in which ComB transports dsDNA across the outer membrane at low force and poor specificity for DNA structure. Subsequently, Hp-ComEC mediates transport into the cytoplasm, leading to the release of the noncovalently bound DNA dye. Taken together, our findings fill the gap to propose a model for composite DNA uptake machineries in competent bacteria. We propose that inner membrane transport is powered by a strong molecular motor that translocates single stranded DNA through the conserved ComEC channel. Various reversible motors that generate low force are responsible for outer membrane transport.
Projektbezogene Publikationen (Auswahl)
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(2019) Type IV pili: dynamics, biophysics and functional consequences, Nat. Rev. Microbiol. 17(7), 429
Craig, L., Forest, K.T., Maier, B.
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(2022) External stresses affect gonococcal type 4 pilus dynamics, Front. Microbiol., 13, 839711
Kraus-Römer, S., Wielert, I., Rathmann, I., Grossbach, J., Maier, B.
