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

Date Published: April 24, 2014

Publisher: Public Library of Science

Author(s): Heike Gangel, Christof Hepp, Stephanie Müller, Enno R. Oldewurtel, Finn Erik Aas, Michael Koomey, Berenike Maier, Gary Dunny.


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.

Partial Text

Natural competence for transformation is widespread among different bacterial species [1]. Transformation is thought to speed up adaptive evolution but it is also discussed in the context of genome maintenance [2][3]. The currently available data from Gram-positive species strongly supports the idea of a coordinated DNA transformation machine that binds DNA at the extracellular side, powers translocation of DNA through the cell envelope and hands the DNA over to the recombination machine at the intracellular side [4][5]. With the only known exception of Helicobacter pylori, all characterized naturally competent species are associated with the type IV pilus (T4P) system for DNA import. At the extracellular side, T4P proteins are essential for DNA binding although it is unclear whether long pilus filaments are necessary [1]. Whereas DNA binding to the competence pilus has been demonstrated in Streptococcus pneumoniae[6], Neisseria gonorrhoeae that generate non-retractile T4P show strongly impaired binding efficiency [7]. In the following, the nomenclature of N. gonorrhoeae will be used to describe the proteins required for transformation. The major pilin subunit is essential for binding and import of DNA [8][7] but replacing the gonococcal pilin PilE by the major subunit of Pseudomonas aeruginosa or of Fracisella tularensis supports DNA import and transformation as well [9], [10]. The transformation rate is modulated by the relative levels of the minor pilins ComP. PilV acts antagonistically at the level of DNA binding with ComP increasing transformability in a dose-dependent fashion and PilV decreasing it [7], [11]. PilV appears to exert its inhibitory effects by competing with ComP for access to the Tfp assembly machinery [11]. The presence of a 12 bp DNA Uptake Sequence (DUS) strongly enhances the probability for DNA-import by N. gonorrhoeae[12][3]. ComP binds DNA in a sequence-specific manner, selecting for DNA containing the DUS [13][7]. The outer membrane channel formed by PilQ is essential for T4P extrusion and DNA import into a DNase-resistant state and moreover shows DNA-binding potential [14], [15]. In the periplasm, three components are linked to transformation. The DNA-binding protein ComE has four identical gene-copies on the gonococcal genome [16]. Gradual deletion of these copies leads to gradual decrease in transformation rate by decreasing the probability for DNA import [16]. The DNA-binding peptidoglycan-linked lipoprotein ComL and the lipoprotein tetrapac (Tpc) which is associated with separation of dividing diplococci are not essential for DNA uptake but for transformation [17][18]. ComA proteins form the channel through which DNA is transported from the periplasm to the cytoplasm [19]. In the Gram-positive species Bacillus subtilis it has been shown that incoming ssDNA is immediately coated by single-strand binding proteins [20]. Single strand binding proteins have been proposed to generate a reservoir of ssDNA in the cytoplasm and to direct the DNA to homologous recombination in B. subtilis and Streptococcus pneumoniae[21][4]. For N. gonorrhoeae there is evidence that ssDNA forms transiently in the periplasm [22].




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