Concerted Spatio-Temporal Dynamics of Imported DNA and ComE DNA Uptake Protein during Gonococcal Transformation

Abstract

Competence for transformation is widespread among bacterial species. In the case of Gram-negative systems, a key step to transformation is the import of DNA across the outer membrane. Although multiple factors are known to affect DNA transport, little is known about the dynamics of DNA import. Here, we characterized the spatio-temporal dynamics of DNA import into the periplasm of Neisseria gonorrhoeae. DNA was imported into the periplasm at random locations around the cell contour. Subsequently, it was recruited at the septum of diplococci at a time scale that increased with DNA length. We found using fluorescent DNA that the periplasm was saturable within minutes with ∼40 kbp DNA. The DNA-binding protein ComE quantitatively governed the carrying capacity of the periplasm in a gene-dosage-dependent fashion. As seen using a fluorescent-tagged derivative protein, ComE was homogeneously distributed in the periplasm in the absence of external DNA. Upon addition of external DNA, ComE was relocalized to form discrete foci colocalized with imported DNA. We conclude that the periplasm can act as a considerable reservoir for imported DNA with ComE governing the amount of DNA stored potentially for transport through the inner membrane.

Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

“…Moreover, previous reports have shown that ComE binds DNA and is essential for DNA uptake (22). In the absence of periplasmic DNA it is homogeneously distributed within the periplasm and relocates to the incoming DNA within minutes (23). The binding energy of ComE to DNA most likely provides the fuel for biasing diffusion of DNA through the secretin in the outer membrane.…”

“…Within the periplasm, the DNAbinding protein ComE is essential for DNA uptake (22). In the absence of transforming DNA, ComE is distributed homogeneously within the periplasm and rapidly colocalizes with imported DNA (23,24). ComE governs the carrying capacity of the periplasm for DNA in a gene-dosage-dependent way (23).…”

“…In the absence of transforming DNA, ComE is distributed homogeneously within the periplasm and rapidly colocalizes with imported DNA (23,24). ComE governs the carrying capacity of the periplasm for DNA in a gene-dosage-dependent way (23). In N. gonorrhoeae and other competent species, transport through the outer membrane is uncoupled from transport through the inner membrane (25)(26)(27).…”

“…We hypothesized that DNA is imported into the periplasm through a translocation ratchet mechanism based on the following data: (i) Uptake of DNA into the periplasm decouples from uptake into the cytoplasm (23). Because ATP is not available in the periplasm, it is unlikely that an ATP-dependent motor drives DNA uptake.…”

“…Similarly, no ion gradient is maintained over the outer membrane. (ii) The periplasmic DNA-binding protein ComE is essential for DNA import into the periplasm (22,23) and depicts an ideal candidate for biasing the direction of DNA movement through the membrane. (iii) Single-cell studies of DNA uptake show that the secondary structure of DNA has only a minor effect on uptake kinetics (30), arguing against a machine that requires a tight fit on its substrate.…”