Single-Stranded DNA Uptake during Gonococcal Transformation

Abstract

Neisseria gonorrhoeae is naturally competent for transformation. The first step of the transformation process is the uptake of DNA from the environment into the cell. This transport step is driven by a powerful molecular machine. Here, we addressed the question whether this machine imports single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) at similar rates. The fluorescence signal associated with the uptake of short DNA fragments labeled with a single fluorescent marker molecule was quantified. We found that ssDNA with a double-stranded DNA uptake sequence (DUS) was taken up with a similar efficiency as dsDNA. Imported ssDNA was degraded rapidly, and the thermonuclease Nuc was required for degradation. In a nuc deletion background, dsDNA and ssDNA with a double-stranded DUS were imported and used as the substrates for transformation, whereas the import and transformation efficiencies of ssDNA with single-stranded DUS were below the detection limits. We conclude that the DNA uptake machine requires a double-stranded DUS for efficient DNA recognition and transports ssDNA and dsDNA with comparable efficiencies. IMPORTANCEBacterial transformation enables bacteria to exchange genetic information. It can speed up adaptive evolution and enhances the potential of DNA repair. The transport of DNA through the outer membrane is the first step of transformation in Gram-negative species. It is driven by a powerful molecular machine whose mechanism remains elusive. Here, we show for Neisseria gonorrhoeae that the machine transports single-and double-stranded DNA at comparable rates, provided that the species-specific DNA uptake sequence is double stranded. Moreover, we found that single-stranded DNA taken up into the periplasm is rapidly degraded by the thermonuclease Nuc. We conclude that the secondary structure of transforming DNA is important for the recognition of self DNA but not for the process of transport through the outer membrane.

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations

“…[28] Second, the motor supports import of dsDNA and single-stranded DNA (ssDNA) with similar kinetics, provided that the DUS is double-stranded. [31] The fact that ComE binds both ssDNA and dsDNA [29] is consistent with this observation. In line with these experiments, ComE(A) comprises a DNA-binding helix-hairpin-helix (HhH) motif that makes contact to DNA backbone phosphates and is thus not sequencespecific.…”

“…First, replacing the native comEA o f V. cholerae by comE of N. gonorrhoeae or even comEA of gram‐positive Bacillus subtilis supported DNA uptake . Second, the motor supports import of dsDNA and single‐stranded DNA (ssDNA) with similar kinetics, provided that the DUS is double‐stranded . The fact that ComE binds both ssDNA and dsDNA is consistent with this observation.…”

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

“…Considering the low dissociation constant of K = 0.0012 provokes the question of how ComE is recycled from the periplasmic DNA. ComE bound to periplasmic DNA is likely to dissociate with time, because DNA is either transported into the cytoplasm or degraded by the thermonuclease Nuc (30). These processes can release ComE, making it available for driving import of new DNA.…”

“…This 12-bp-long sequence occurs at high frequency on the gonococcal genome, conveying species-specific recognition of DNA. Double-stranded but not single-stranded DUS enhances the probability of DNA uptake (30). The minor pilin ComP is responsible for binding the DUS and DNA import is Significance Transport of macromolecules through nanometer-sized membrane pores is a ubiquitous theme in cell biology.…”

“…(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. Here, we characterized the velocity-force relation of single-DNA import and found that it is consistent with the model of a translocation ratchet.…”