Single-Stranded DNA Uptake during Gonococcal Transformation.

UNLABELLED: 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. IMPORTANCE: Bacterial 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.

Differential interaction forces govern bacterial sorting in early biofilms.

Bacterial biofilms can generate micro-heterogeneity in terms of surface structures. However, little is known about the associated changes in the physics of cell-cell interaction and its impact on the architecture of biofilms. In this study, we used the type IV pilus of Neisseria gonorrhoeae to test whether variation of surface structures induces cell-sorting. We show that the rupture forces between pili are fine-tuned by post-translational modification. Bacterial sorting was dependent on pilus post-translational modification and pilus density. Active force generation was necessary for defined morphologies of mixed microcolonies. The observed morphotypes were in remarkable agreement with the differential strength of adhesion hypothesis proposing that a tug-of-war among surface structures of different cells governs cell sorting. We conclude that in early biofilms the density and rupture force of bacterial surface structures can trigger cell sorting based on similar physical principles as in developing embryos.

A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity.

Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.